TW202023582A - Oncolytic virus platform to treat hematological cancer - Google Patents

Abstract

The disclosure provides Myxoma virus that expresses one or more immunomodulatory transgenes and its use in inhibiting and/or treating a hematological cancer in a subject. The disclosure also provides a leukocyte having a Myxoma virus that expresses one or more immunomodulatory transgenes and the use of the leukocyte for inhibiting and/or treating a hematological cancer in a subject.

Description

Oncolytic virus platform for the treatment of hematological cancers

The present invention relates to myxoma virus and its use for the treatment of cancer, for example, the myxoma virus expressing one or more immunomodulatory transgenic genes (such as BiKE, LIGHT and/or Decorin) is used to treat hematological cancer.

The current treatment methods used to treat various types of cancer tend to work by poisoning or killing cancer cells. Unfortunately, treatments that are toxic to cancer cells also tend to be toxic to healthy cells. In addition, effective treatment of cancer is still difficult to achieve. Current mainstream therapies such as chemotherapy and radiotherapy may have a narrow therapeutic window (for example, a concentration sufficient to obtain efficacy but low enough to avoid toxicity). These types of therapies are regarded as blunt tools with limited applicability due to the different types of tumor cells and the limited window available for administration of these treatment methods.

In some aspects, this paper discloses a myxoma virus (MYXV) containing one or more immunomodulatory transgenic genes, wherein the one or more immunomodulatory transgenic genes encode BiKE (bispecific natural killer and neutrophil Leukocyte junction molecules), LIGHT (lymphotoxins, presenting inducible expression, and competing with HSV glycoprotein D for herpes virus entry mediator (HVEM)-a receptor expressed by T lymphocytes), Decorin or a combination thereof.

In some embodiments, the myxoma virus comprises MYXV-BiKE. In some embodiments, BiKE binds to antigens present on natural killer cells, neutrophils, or combinations thereof. In some embodiments, BiKE binds to antigens present on blood cancer cells. In some embodiments, BiKE binds to an antigen present on myeloma cells. In some embodiments, BiKE binds to an antigen present on leukemia cells. In some embodiments, BiKE binds to an antigen present on lymphoma cells. In some embodiments, BiKE binds to CD16. In some embodiments, BiKE binds CD138. In some embodiments, BiKE comprises one or more single chain variable fragments (scFv). In some embodiments, BiKE comprises one or more humanized single chain variable fragments (scFv). In some embodiments, BiKE comprises a sequence that is at least 70% identical to any of SEQ ID NO: 6 or 16-31. In some embodiments, BiKE is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-huBiKE-GFP. In some embodiments, the myxoma virus comprises MYXV-LIGHT. In some embodiments, LIGHT comprises a sequence from human LIGHT. In some embodiments, LIGHT comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 13-15. In some embodiments, LIGHT is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-FLuc-huLIGHT-TdTomato. In some embodiments, the myxoma virus comprises MYXV-Decorin. In some embodiments, Decorin comprises a sequence from human Decorin. In some embodiments, Decorin comprises a sequence from mouse Decorin. In some embodiments, Decorin comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 7-12. In some embodiments, Decorin is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-mDecorin-GFP. In some embodiments, the myxoma virus further includes a reporter gene. In some embodiments, the reporter gene is a fluorescent protein. In some embodiments, the reporter gene is a luminescent substrate or an enzyme. In some embodiments, the myxoma virus further comprises a deletion in the myxoma virus genome. In some embodiments, the myxoma virus includes deletion or destruction of one or more genes selected from the group consisting of: M001R, M002R, M003.1R, M003.2R, M004.1R, M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2, M-T4, M-T5, M-T7 and SOD. In some embodiments, the myxoma virus comprises a deletion of M135. In some embodiments, the myxoma virus is part of a composition comprising myxoma virus and a pharmaceutically acceptable carrier.

In some aspects, this document discloses a method of treating hematological cancer in an individual in need, which comprises administering to the individual a myxoma virus containing one or more immunomodulatory transgenic genes.

In some embodiments, the one or more immunomodulatory transgenic genes comprise BiKE, LIGHT, Decorin, or a combination thereof. In some embodiments, the myxoma virus comprises MYXV-BiKE. In some embodiments, BiKE binds to antigens present on natural killer cells, neutrophils, or combinations thereof. In some embodiments, BiKE binds to antigens present on blood cancer cells. In some embodiments, BiKE binds to an antigen present on myeloma cells. In some embodiments, BiKE binds to an antigen present on leukemia cells. In some embodiments, BiKE binds to an antigen present on lymphoma cells. In some embodiments, BiKE binds to CD16. In some embodiments, BiKE binds CD138. In some embodiments, BiKE comprises one or more single chain variable fragments (scFv). In some embodiments, BiKE comprises one or more humanized single chain variable fragments (scFv). In some embodiments, BiKE comprises a sequence that is at least 70% identical to any of SEQ ID NO: 6 or 16-31. In some embodiments, BiKE is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-huBiKE-GFP. In some embodiments, the myxoma virus comprises MYXV-LIGHT. In some embodiments, LIGHT comprises a sequence from human LIGHT. In some embodiments, LIGHT comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 13-15. In some embodiments, LIGHT is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-FLuc-huLIGHT-TdTomato. In some embodiments, the myxoma virus comprises MYXV-Decorin. In some embodiments, Decorin comprises a sequence from human Decorin. In some embodiments, Decorin comprises a sequence from mouse Decorin. In some embodiments, Decorin comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 7-12. In some embodiments, Decorin is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-mDecorin-GFP. In some embodiments, the myxoma virus further includes a reporter gene. In some embodiments, the reporter gene is a fluorescent protein. In some embodiments, the reporter gene is a luminescent substrate or an enzyme. In some embodiments, the myxoma virus further comprises a deletion in the myxoma virus genome. In some embodiments, the myxoma virus includes deletion or destruction of one or more genes selected from the group consisting of: M001R, M002R, M003.1R, M003.2R, M004.1R, M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2, M-T4, M-T5, M-T7 and SOD. In some embodiments, the myxoma virus comprises a deletion of M135. In some embodiments, the individual is a human. In some embodiments, the myxovirus can infect cells with defective innate antiviral responses. In some embodiments, the myxoma virus can infect cancer cells. In some embodiments, the blood cancer is myeloma, leukemia, or lymphoma. In some embodiments, the blood cancer is multiple myeloma.

In some aspects, this document discloses a method of treating hematological cancer in an individual in need, which comprises administering to the individual a white blood cell, wherein the white blood cell contains a myxoma virus containing one or more immunomodulatory transgenic genes.

In some embodiments, the method further comprises adsorbing myxoma virus to the surface of white blood cells ex vivo. In some embodiments, adsorbing the myxoma virus onto the surface of the white blood cell includes exposing the white blood cell to the myxoma virus under conditions that permit the myxoma virus to bind to the surface of the white blood cell. In some embodiments, the myxoma virus is exposed to white blood cells for at least five minutes. In some embodiments, the myxoma virus is exposed to white blood cells for about one hour. In some embodiments, the myxoma virus is exposed to white blood cells at a multiplicity of infection (MOI) between about 0.001 and 1000. In some embodiments, the myxoma virus is exposed to white blood cells at a magnification of infection (MOI) between about 0.1 and 10. In some embodiments, the white blood cells are obtained from peripheral blood. In some embodiments, the white blood cells are obtained from bone marrow. In some embodiments, the white blood cells are peripheral blood mononuclear cells. In some embodiments, the white blood cells are obtained from an individual. In some embodiments, the white blood cells are obtained from a donor that is HLA-matched, HLA-mismatched, haploid-matched, or a combination thereof relative to the individual. In some embodiments, the one or more immunomodulatory transgenic genes comprise BiKE, LIGHT, Decorin, or a combination thereof. In some embodiments, the myxoma virus comprises MYXV-BiKE. In some embodiments, BiKE binds to antigens present on natural killer cells, neutrophils, or combinations thereof. In some embodiments, BiKE binds to antigens present on blood cancer cells. In some embodiments, BiKE binds to an antigen present on myeloma cells, leukemia cells, or lymphoma cells. In some embodiments, BiKE binds to CD16. In some embodiments, BiKE binds CD138. In some embodiments, BiKE comprises one or more single chain variable fragments (scFv). In some embodiments, BiKE comprises one or more humanized single chain variable fragments (scFv). In some embodiments, BiKE comprises a sequence that is at least 70% identical to any of SEQ ID NO: 6 or 16-31. In some embodiments, BiKE is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-huBiKE-GFP. In some embodiments, the myxoma virus comprises MYXV-LIGHT. In some embodiments, LIGHT comprises a sequence from human LIGHT. In some embodiments, LIGHT comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 13-15. In some embodiments, LIGHT is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-FLuc-huLIGHT-TdTomato. In some embodiments, the myxoma virus comprises MYXV-Decorin. In some embodiments, Decorin comprises a sequence from human Decorin. In some embodiments, Decorin comprises a sequence from mouse Decorin. In some embodiments, Decorin comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 7-12. In some embodiments, Decorin is between the M135 and M136 open reading frames of the myxoma virus genome. In some embodiments, the myxoma virus comprises MYXV-mDecorin-GFP. In some embodiments, the myxoma virus further includes a reporter gene. In some embodiments, the reporter gene is a fluorescent protein. In some embodiments, the reporter gene is a luminescent substrate or an enzyme. In some embodiments, the myxoma virus further comprises a deletion in the myxoma virus genome. In some embodiments, the myxoma virus includes deletion or destruction of one or more genes selected from the group consisting of: M001R, M002R, M003.1R, M003.2R, M004.1R, M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2, M-T4, M-T5, M-T7 and SOD. In some embodiments, the myxoma virus comprises a deletion of M135. In some embodiments, the individual is a human individual. In some embodiments, the individual has or is suspected of having cancer. In some embodiments, the myxovirus can infect cells with defective innate antiviral responses. In some embodiments, the blood cancer is myeloma, leukemia, or lymphoma. In some embodiments, the blood cancer is multiple myeloma. In some embodiments, the white blood cells are administered as a pharmaceutical composition. In some embodiments, the white blood cells are administered systemically. In some embodiments, the white blood cells are administered parenterally. In some embodiments, the white blood cells are administered by infusion.

cross reference

This application claims the rights and interests of U.S. Provisional Application No. 62/727,307 filed on September 5, 2018, which is incorporated herein by reference.

The aspect of the present invention relates to an oncolytic virus recombinant construct expressing an immunomodulatory transgene and its use in the treatment of cancer (such as blood cancer). The oncolytic virus may be myxoma virus (MYXV), and the immunomodulatory transgenes used in the construct may include Decorin transgenes, BiKE (bispecific natural killer and neutrophil conjugating molecule) transgenes, LIGHT (lymphotoxins, presents inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM)-a receptor expressed by T lymphocytes) transgenic genes or combinations thereof. The MYXV described herein can be used to treat hematological cancers, including minimal residual disease (MRD) and drug-resistant MRD.

The MYXV described herein can be a more effective therapy for treating hematological cancers (such as recurrent multiple myeloma disease) and completely eliminating refractory and drug-resistant MRDs. Take multiple myeloma (MM) as an example. MM is a hematological malignancy characterized by asexual expansion of malignant plasma cells, causing terminal organ damage, including osteolytic lesions, anemia, renal failure or hypercalcemia ( Hari P. Recent advances in understanding multiple myeloma. Hematol Oncol Stem Cell Ther. 2017; to be published). MM bone marrow (BM) tumor microenvironment plays a key role in supporting and maintaining the differentiation, migration, proliferation, survival and drug resistance of malignant MM cells (Kawano Y, Moschetta M, Manier S, Glavey S, Görgün GT, Roccaro AM Et al. Targeting the bone marrow microenvironment in multiple myeloma. Immunol Rev. 2015;263(1)). Autologous stem cell transplantation and chemotherapy for qualified patients for transplantation are the standard treatment of MM (Landgren O, Lu SX and Hultcrantz M. MRD Testing in Multiple Myeloma: The Main Future Driver for Modern Tailored Treatment. Semin Hematol. 2018; 55( 1):44-50; Hoyos V and IB The immunotherapy era of myeloma: monoclonal antibodies, vaccines, and adoptive T-cell therapies. Blood. 2016;128(13):1679-87). However, the main obstacle of these therapies is the recurrence of the disease due to the neoplastic clone that can serve as a reservoir for the treatment of resistant MM cells, causing minimal residual disease (MRD).

Despite the improved results, MM is still considered incurable for most patients, and poor survival rates have been observed in patients with high-risk characteristics. (Bustoros M, Mouhieddine TH, Detappe A and IM. G. Established and Novel Prognostic Biomarkers in Multiple Myeloma. Am Soc Clin Oncol Educ Book. 2017;37:548-60). Oncolytic viruses (such as MYXV) are mammalian viruses that can be designed and/or selected for their ability to selectively infect and kill transformed cancer cells and their ability to activate the host immune system. The MYXV described herein utilizes immunomodulatory transgenic genes and can be combined with the host immune system to target cancer cells. Therefore, the myxoma virus described herein can help reduce or eliminate refractory and drug-resistant minimal residual diseases, and can more effectively treat recurrent MM diseases. definition

Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCR Publishers, Inc., 1995 (ISBN 1-56081- 569-8).

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The following explanations of terms and methods are provided to better describe the compounds, compositions and methods of the present invention, and to guide those who are generally familiar with the art in the practice of the present invention. It should also be understood that the terms used in the present invention are only used for the purpose of describing the advantages of specific embodiments and examples and are not intended to be limited.

As used herein, unless the context clearly dictates otherwise, the singular forms "a" and "the" are intended to also include the plural forms.

As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the listed related items, as well as lack of combinations when interpreted as alternatives ("or").

As used herein, "one or more (species)" or at least one can mean one, two, three, four, five, six, seven, eight, nine, ten, or ten Above up to any number.

As used herein, the term "comprises/comprising" means "comprising." Therefore, "including A or B" means including A, B or A and B. The terms "comprise" and variations (such as "comprising (comprising, comprises, and comprised)") as used herein mean that various additional components or steps can be used in combination.

"Effective amount" or "therapeutically effective amount" refers to the amount of the compound or composition of the present invention that is sufficient to produce the desired effect, which can be therapeutic and/or beneficial. In this example, the effective amount can vary with the following: age, general condition of the individual, severity of the condition being treated, specific agent administered, duration of treatment, nature of any simultaneous treatment, and the type of treatment used Pharmaceutically acceptable carriers and similar factors in the knowledge and expertise of those familiar with the technology. When appropriate, the effective amount or therapeutically effective amount in any individual case can be determined by referring to relevant texts and literature and/or by experiments. (See, for example, Remington, The Science and Practice of Pharmacy (latest edition)).

As used herein, the terms "individual" and "patient" are used interchangeably and refer to both human and non-human animals. The term "non-human animal" in the present invention includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, rodents (such as mice, rats, etc.) ) And its analogs. The individual may be a human. The individual may be a human patient. In some embodiments, the subject of the present invention is a human subject.

The term "cell" as used herein includes a single cell as well as a plurality of cells or cell populations. Administration of the agent or exposure of the agent to cells may include in vitro, ex vivo, and in vivo administration or exposure.

"Individuals in need" or "individuals in need" are individuals who are known to have or are suspected of having cancer (such as blood cancer).

As used herein, the term "cancer" refers to a malignant neoplasm, for example, a malignant neoplasm that has undergone characteristic degeneration with loss of differentiation, increased growth rate, invasion of surrounding tissues, and capable of cancer metastasis.

Residual cancer is cancer that remains in the individual after any form of treatment provided to the individual to reduce or eradicate the cancer. Metastatic cancer is cancer at one or more parts of the body (for example, a second part other than the original (primary) cancer where the metastatic cancer originated). Local recurrence is the recurrence of a cancer that is at or near the same site (such as in the same tissue) as the original cancer. Hematological cancers are cancers that affect the blood, bone marrow, and/or lymphatic system.

Non-limiting examples of hematological cancers include leukemia, lymphoma, and myeloma, such as: multiple myeloma (MM); active multiple myeloma; smoldering multiple myeloma; plasmacytoma; solitary bone plasma cell Tumor; Extramedullary plasmacytoma; Light chain myeloma; Nonsecretory myeloma; Immunoglobulin G (IgG) myeloma; Immunoglobulin A (IgA) myeloma; Immunoglobulin M (IgM) myeloma; Immunity Globulin D (IgD) myeloma; Immunoglobulin E (IgE) myeloma; Hyperdiploid multiple myeloma; Non-hyperdiploid multiple myeloma; Hodgkin lymphoma (Hodgkin lymphoma); non Hodgkin's lymphoma; acute lymphoblastic leukemia; acute myeloid leukemia; essential thrombocythemia; polycythemia vera; primary myelofibrosis; systemic mastocytosis; chronic myelogenous leukemia; chronic Neutrophilic leukemia; chronic eosinophilic leukemia; refractory anemia with ring-shaped iron-containing embryo blood cells; refractory cytopenia with multi-lineage dysplasia; refractory anemia with excessive blast type 1; excessive mother Refractory anemia of cell type 2; Myelodysplastic syndrome (MDS) with isolated del (5q); Unclassified MDS; Chronic myelomonocytic leukemia (CML); Atypical chronic myelogenous leukemia; Juvenile bone marrow Monocytic leukemia; myeloproliferative/myelodysplastic syndrome-not classified; B lymphoblastic leukemia/lymphoma; T lymphoblastic leukemia/lymphoma; diffuse large B-cell lymphoma; primary central nervous system Lymphoma; primary mediastinal B-cell lymphoma; Burkitt lymphoma/leukemia; follicular lymphoma; chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma; B-cell prelymphoma Leukemia; lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia; mantle cell lymphoma; marginal zone lymphoma; lymphoma after transplantation; HIV-related lymphoma; primary Exudative lymphoma; intravascular large B-cell lymphoma; primary skin primary skin B-cell lymphoma; hairy cell leukemia; gamma globulin disease of unknown significance; multiform large cell lymphoma, vascular Immunoblastic T cell lymphoma, hepatocellular splenic T cell lymphoma, B cell lymphoma, reticuloendothelial cell hyperplasia, reticulocytosis, mucosa-associated lymphoid tissue lymphoma, B cell chronic lymphocytic leukemia, Waldenstrom's macroglobulinemia, lymphoma-like granuloma, Hodgkin's lymphoma with prominent nodular lymphocytes, plasma cell leukemia, acute polycythemia and erythroleukemia, erythremic myelosis , Acute red blood cell leukemia, Heilmeyer-Schöner disease, acute megakaryoblastic leukemia, Mast cell leukemia, panmyelopathy, acute panmyelopathy with myelofibrosis, lymphosarcoma cell leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, subacute leukemia of unspecified cell type, accelerated chronic myeloid Leukemia, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute monocytic leukemia, mature acute myeloid leukemia, acute bone marrow dendritic leukemia, adult blastic T cell leukemia/lymph Tumor, aggressive NK-cell leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic idiopathic myelofibrosis, Kahler's disease, myeloma disease, solitary myeloma , Plasma cell leukemia, vascular central immunoproliferative disease, lymphoid granuloma, angioimmunoblastic lymphadenopathy, T-γ lymphoproliferative disease, Waldenstrom macroglobulinemia, alpha heavy chain disease, γ heavy chain disease and Franklin's disease (Franklin's disease). In some embodiments, the blood cancer is multiple myeloma.

As used herein, the term "chemotherapeutic agent" refers to any chemical agent that has therapeutic effectiveness in treating diseases characterized by abnormal cell growth. Such diseases can include tumors, neoplasms, and cancers, as well as diseases characterized by proliferative growth such as psoriasis. In some embodiments, the chemotherapeutic agent is an agent that treats cancer, such as an anti-tumor agent. In some embodiments, the chemotherapeutic agent is a radioactive compound. Those familiar with this technique can easily identify the chemotherapeutics used (see, for example, Slapak and Kufe, Principles of Cancer Therapy , Harrison's Principles of Internal Medicine , Chapter 86, 14th edition; Perry et al., Chemotherapy , Abeloff, Chapter 17 of Clinical Oncology , 2nd Edition, 2000 Churchill Livingstone, Inc; Baltzer and Berkery. (eds): Oncology Pocket Guide to Chemotherapy , 2nd Edition St. Louis, Mosby-Year Book, 1995; Fischer Knobf and Durivage (eds) ): The Cancer Chemotherapy Handbook , 4th edition St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one therapeutic agent to treat cancer. For example, a myxoma virus expressing an immunomodulatory transgene can be administered, and one or more chemotherapeutic agents can be administered simultaneously or separately in any order at appropriate times.

As used herein, "treat/treatment/treating" refers to the administration of a pharmaceutical composition to a patient suffering from a disease or condition. As used herein, the term "inhibition" or "treatment of a disease", such as cancer, refers to delaying or inhibiting the development or progression of a disease or condition. "Treatment" refers to therapeutic intervention to improve the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "improvement" in relation to a disease or pathological condition means that any observable therapeutic effect is beneficial. It can be, for example, delayed by the onset of clinical symptoms in susceptible individuals, reduced severity of some or all of the clinical symptoms of the disease, slower progression of the disease (such as cancer metastasis), improvement in the overall health or well-being of the individual, or by a specific disease. Other parameters that are unique to this technology proved beneficial. "Prophylactic" treatment is for the purpose of reducing the risk of developing disease, such as metastatic cancer, to administer treatment to individuals who do not show symptoms of the disease or only show early symptoms of the disease.

As used herein, "pharmaceutically acceptable carriers" suitable for use in combination with the therapeutic compounds disclosed herein may be conventional. Remington 's Pharmaceutical Sciences, by the EW Martin, Mack Publishing Co., Easton , Pa. The, 19th Edition (1995), describe pharmaceutical agent suitable for the delivery of therapeutic compositions and formulations.

In general, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations typically include injectable fluids including pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous glucose solutions, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, lozenge or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents and the like, such as sodium acetate or sorbitan monolaurin Acid ester.

As used herein, the terms "medicine" and "therapeutic agent" refer to compounds or compositions that can induce a desired therapeutic or preventive effect when properly administered to an individual or cell.

As used herein, the term "replicable" refers to the ability of viruses (such as myxoma virus) to infect and replicate in specific host cells (such as human blood cells (eg, peripheral blood mononuclear cells)).

The term "immunomodulatory transgene" refers to a gene sequence that can be introduced into the viral genome and encodes a product that can affect the function of the immune system, such as affecting inflammation, innate or adaptive immune signaling, congenital or adaptive Immune cell activation (e.g., target cell killing, production of cytokines, chemokines, or other inflammatory mediators), innate or adaptive immune cell homing (e.g., chemotaxis, extravasation, and / Or accumulation), or innate or adaptive immune cell proliferation, innate or adaptive immune cell differentiation, antibody production, or a combination thereof. Examples of immunomodulatory transgenes include, but are not limited to, Decorin, BiKE, and LIGHT. Myxoma virus

Myxoma virus (MYXV) is potentially very suitable as a therapeutic virus for blood cancers, such as multiple myeloma (MM) due to its unique biology. MYXV is a member of the genus Poxvirus and Harepoxvirus (Chan WM, Rahman MM and McFadden G. Oncolytic myxoma virus: the path to clinic. Vaccine. 2013;31(39):4252-8, Chan WM and McFadden G. Oncolytic Poxviruses. Annu Rev Virol. 2014;1(1):119-41).

MYXV is a novel oncolytic virus (Stanford MM and McFadden G. Myxoma virus and oncolytic virotherapy: a new biologic weapon in the war against cancer) that can target various human and murine cancers (both primary and defined cell lines) . Expert Opin Biol Ther. 2007;7(9):1415-11425; Wang G, Barrett JW, Stanford M, Werden SJ, Johnston JB, Gao X et al. Infection of human cancer cells with myxoma virus requires Akt activation via interaction with a viral ankyrin-repeat host range factor. Proc Natl Acad Sci USA. 2006;103(12):4640-5; Bartee E, Chan WM, Moreb JS, Cogle CR and McFadden G. Selective purging of human multiple myeloma cells from autologous stem cell transplantation grafts using oncolytic myxoma virus. Biol Blood Marrow Transplant. 2012;18(10):1540-51; Chan WM, Rahman MM and McFadden G. Oncolytic myxoma virus: the path to clinic. Vaccine. 2013;31(39 ): 4252-8; Kim M, Madlambayan GJ, Rahman MM, Smallwood SE, Meacham AM, Hosaka K, et al. Myxoma virus targets primary human leukemic stem and progenitor cells while sparing normal hematopoitic stem and progenitor cells. Leukemia. 2009; 32: 2313- 7; Villa NY, Wasserfall CH, Meacham AM, Wise E, Chan W, Wingard JR et al. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells. Blood. 2015;125(24):3778-88 ).

In essence, MYXV is unique to rabbits only and does not cause infection or disease in humans, mice or any other domestic animals. However, due to the nature of cancer pathway mutations associated with carcinogenesis, cancer cells from both mice and humans may exhibit impaired resistance to infection by some viruses (including MYXV) (such as impaired innate immune pathways). (Chan WM and McFadden G. Oncolytic Poxviruses. Annu Rev Virol. 2014;1(1):119-41, Sypula J,'Wang F, Ma Y, Bell J and McFadden G. Myxoma virus tropism in human tumors. Gene Ther and Mol Biol. 2004;8:103-14).

In some embodiments, provided herein is a modified myxoma virus (MYXV). MYXV can be any virus belonging to a species of harepox virus that can replicate. MYXV may be a wild-type strain of MYXV or it may be a genetically modified strain of MYXV. In some cases, MYXV is a Lausanne virus strain. In some cases, MYXV is a South American MYXV strain circulating in South American forest rabbits (Sylvilagus brasiliensis). In some cases, MYXV is a California strain of MYXV circulating in Sylvilagus bachmani. In some cases, MYXV is 6918, an attenuated Spanish domain virus strain, which contains genes M009L, M036L, M135R and M148R (Genbank accession number EU552530, which is hereby incorporated by reference, as GenBank in August 2019 Provided on the 27th) modification. In some cases, MYXV is 6918VP60-T2 (GenBank deposit number EU552531, which is hereby incorporated by reference, as provided by GenBank on August 27, 2019). In some cases, MYXV is SG33, a virus strain containing a genome deletion that affects genes M151R, M152R, M153R, M154L, M156R, M008.1R, M008R, M007R, M006R, M005R, M004.1R, M004R , M003.2R, M003.1R, M002R and M001R (Collection Nationale de Cultures de Microorganismes (CNCM) Accession No. 1-1594). In some cases, MYXV is a virus strain called Standard laboratory Strain (SLS).

In some cases, MYXV contains at least 70%, 75%, 80%, 85% of the sequence disclosed in Cameron et al., "The complete DNA sequence of Myxoma Virus," Virology 264: 298-318 (1999) , 90%, 95%, 96%, 97%, 98% or 99%, such as between 95% and 98%, 95% and 99%, including 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% sequence identity. In some cases, MYXV contains the sequence disclosed in Cameron et al., "The complete DNA sequence of Myxoma Virus," Virology 264: 298-318 (1999).

Larger and genetically stable poxvirus genomes can be genetically manipulated, for example to produce viruses with one or more deletions and/or introduction of one or more therapeutic (e.g., immunomodulatory) transgenic genes (Nayerossadat N, Maedeh T and Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res. 2012;1:27).

In some embodiments, provided herein is myxoma virus (MYXV) and modified MYXV. MYXV can be any virus belonging to a species of harepox virus that can replicate. MYXV may be a wild-type strain of MYXV or it may be a genetically modified strain of MYXV.

The myxoma virus genome can be modified using molecular biology techniques known to those skilled in the art and described in, for example, Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual, 3rd Edition, Cold Spring Harbour Laboratory Press) Express one or more therapeutic transgenes (e.g., immunomodulatory transgenes such as BiKE, LIGHT, and/or Decorin). Those who are familiar with this technology will be able to determine which parts of the myxovirus genome can be deleted so that the virus can still effectively infect, for example, to provide a replicable virus. For example, non-essential regions of the viral genome that can be deleted can be derived from comparing published viral genome sequences with other well-characterized viral genomes (see, for example, C. Cameron, S. Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay, D. Willer, D. Evans and G. McFadden, Virology (1999) 264: 298-318)).

In some embodiments, the disclosed MYXV recombinant constructs are used to treat relapsed/refractory primary human hematological malignancies such as multiple myeloma (MM) and to target and reduce or eliminate minimal residual disease (MRD) Oncolytic virus candidates. In some embodiments, MYXV contains one or more transgenic genes.

In some embodiments, the MYXV of the present invention includes one or more genetic modifications, deletions and/or disruptions in the MYXV genome. For example, the MYXV of the present invention may include one or more gene insertions, deletions, or substitutions within or adjacent to one or more genes in the genome. Gene insertion, deletion, or modification may include gene deletion (e.g., deletion of one or more nucleotides to hinder the functionality of the product encoded by the gene or insertion of one or more nucleotides to disrupt the performance of the product encoded by the gene and / Or function). In some embodiments, the insertion, deletion, or modification does not include gene knockout (for example, a sequence can be inserted at an intergenic locus between two genes without disrupting the performance of the two genes).

In some embodiments, the MYXV of the present invention includes one or more gene insertions, deletions, or substitutions in or near one or more genes, and the one or more genes are related to the virus's ability to cause host animal diseases. In some embodiments, the MYXV of the present invention includes one or more gene insertions, deletions, or substitutions in or near one or more genes related to host cell tropism. In some embodiments, the MYXV of the present invention includes one or more gene insertions, deletions, or substitutions within or near one or more genes, and the one or more genes are related to the virus's ability to evade innate immune responses. In some embodiments, the MYXV of the present invention comprises one or more insertions, deletions or substitutions in or near one or more genes that regulate the immune signal transduction of infected cells (such as interleukin receptors). Body signaling). In some embodiments, the MYXV of the present invention comprises one or more gene insertions, deletions or substitutions within or near one or more genes, and the one or more genes regulate the cell death pathway of infected cells (for example, codes that promote Or a gene that inhibits the product of apoptosis, such as M011L). In some embodiments, the MYXV of the present invention comprises one or more insertions, deletions or substitutions within or near one or more genes that regulate viral replication in cancer cells (for example, increase or decrease cancer The rate of virus replication in the cell).

In some embodiments, one or more genes related to the ability of the virus to cause disease in the host animal, genes related to the tropism of the host cell, and genes related to the ability of the virus to evade innate immune responses can regulate the infecting cells The immune signal transduction gene, the gene that can regulate the cell death pathway in the infected cell, the gene that can regulate the virus replication in cancer cells, or the combination of genes, including any one or more of the following: M001R, M002R, M003.1R, M003.2R, M004.1R, M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2, M-T4, M-T5, M-T7 and SOD.

In some embodiments, the MYXV of the present invention includes a modification of the MYXV gene. In some cases, the modification is a deletion that impairs the function of the protein encoded by the MYXV gene. In some cases, the modification is a partial deletion of the MYXV gene (eg, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% deletion). In other cases, the modification is a complete deletion of the MYXV gene. In some embodiments, the modification is to replace the MYXV gene with one or more transgenic genes of the present invention (for example, BiKE, Decorin, and/or LIGHT).

In some embodiments, the MYXV of the present invention includes one or more insertions, deletions, or substitutions in or near one or more genes related to host cell tropism (for example, rabbit cell tropism). In some embodiments, the one or more genes associated with rabbit cell tropism include M11L, M063, M135R, M136R, M-T2, M-T4, M-T5, M-T7, or a combination thereof. In some cases, the one or more genes associated with rabbit cell tropism include M135R, M136R, or a combination thereof.

In some embodiments, the MYXV of the present invention includes a modification of the M135R gene. In some embodiments, MYXV includes a partial or complete deletion of the M135R gene. Deletion or destruction of the M135R gene can, for example, weaken the ability of MYXV of the present invention to cause disease in the host animal without compromising the ability of MYXV to exhibit anti-cancer effects (such as infection and killing cancer cells). In some cases, the modification is a deletion that impairs the function of the protein encoded by the M135R gene. In some cases, the modification is a partial deletion of the M135R gene (eg, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% deletion). In other cases, the modification is a complete deletion of the M135R gene. In some embodiments, MYXV includes a modification of the M135R gene that impairs the function of the M135R gene (for example, inserting a sequence that disrupts the expression and/or function of the M135R gene). In some embodiments, the transgenic gene of the present invention replaces the M135R gene in the MYXV genome (for example, one or more transgenic genes of the present invention (such as BiKE, Decorin and/or LIGHT) are used to destroy or replace the M135R gene). In some embodiments, the transgenic gene of the present invention is inserted between the M135R gene and the M136R gene in the MYXV genome. Transgenic

In some embodiments, provided herein is a myxoma virus (MYXV) recombinant construct containing a transgenic gene.

In the case of cancer and tumor microenvironment, a series of immunomodulatory factors can affect the interaction between cancer cells and the immune system. For example, immunomodulatory factors can directly interact with malignant cells, positively or negatively affect the infiltration of cytotoxic lymphocytes into the tumor microenvironment, and positively or negatively affect the cell-mediated immune response to cancer cells. The production of chemokines and chemokines, or their combination. One or more immunomodulatory transgenic genes can be introduced into the MYXV genome, for example, to promote more effective treatment or reduce the immune response to cancer.

For example, in some embodiments, one or more MYXV endogenous genes that regulate different forms of immune regulation and/or cell death are removed. In some embodiments, one or more therapeutic immunomodulatory transgenic genes are introduced into the viral genome (e.g., in a preferred form to increase immunogenicity and/or induce cancer cell death). In some embodiments, one or more MYXV endogenous genes are removed, and one or more immunomodulatory transgenic genes are introduced into the viral genome.

In some embodiments, the transgenic gene is an immunomodulatory transgenic gene. In some embodiments, MYXV of the present invention includes Decorin transgenic gene, BiKE (bispecific natural killer and neutrophil conjugating molecule) transgenic gene, LIGHT (corresponding to lymphotoxin, exhibits inducible expression and is compatible with HSV sugar Protein D competes with herpes virus entry mediator (HVEM)-a receptor expressed by T lymphocytes) for gene transfer or a combination thereof.

The MYXV of the present invention may include Decorin transgenic gene (MYXV-Decorin). Decorin is an example of an immunomodulatory transgenic gene that can be introduced into the MYXV genome. Decorin is a small leucine-rich proteoglycan that exhibits tumor suppressing properties. For example, decorin can bind to and inhibit TGF-β, thereby alleviating immunosuppression in the tumor microenvironment. Compared with healthy volunteers, patients with MM produced lower levels of decorin (Nemani N, Santo L, Eda H, Cirstea D, Mishima Y, Patel C, et al. Role of decorin in multiple myeloma (MM) bone marrow microenvironment. J Bone Miner Res. 2015;30(3):465-70).

In some embodiments, the Decorin transgenic gene comprises a sequence from a mammalian Decorin gene. In some embodiments, the Decorin transgenic gene comprises a sequence from the mouse Decorin gene (mDecorin). In some embodiments, the Decorin transgenic gene comprises a sequence from the human Decorin gene (huDecorin). In some embodiments, the Decorin transgenic gene encodes a secreted product. In some embodiments, the Decorin transgenic gene encodes a product that is localized to the cell surface (eg, contains a transmembrane domain). As provided in Table 1, in some embodiments, from of Decorin gene comprises SEQ ID NO: 7 - 12 of the sequence of any one of. Table 1 SEQ ID NO: description sequence 7 Human Decorin, homomorphic A MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQELRAHENEITKVRKVTFNGLNQMIVIELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYK 8 Human Decorin, homomorphic B MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLHLDNNKLTRVVRCVAEGSLANTPHLDNNKLTRVVRCVAEGSLANTPHLHLDNNKLTRVVRCVAEGSLANTPHLDNNKLTRVVRCVAEGSLANTPHLDNNKLTRVVVAEGSLANTPHLDNNKLTRVVVCVAEGSLFGSYSPVVAEQ 9 Human Decorin, homomorphic C MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVVYLHNNNISVVGSSDFCPPGHNTKRCKASYSVRSAVVYLHNNNISQCHLRVVQCSDLGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPGHNTKRCKASYSVRSAVVVYLHNNNISVVGSSDFCPPGHNTKRCKASYSVYVSRSQVYV 10 Human Decorin, homomorphic D MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHVVYLHNNNISVVGSSDFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLG 11 Human Decorin, homomorphic E MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGCLPS 12 Mouse Decorin MKATLIFFLLAQVSWAGPFEQRGLFDFMLEDEASGIIPYDPDNPLISMCPYRCQCHLRVVQCSDLGLDKVPWDFPPDTTLLDLQNNKITEIKEGAFKNLKDLHTLILVNNKISKISPEAFKPLVKLERLYLSKNQLKELPEKMPRTLQELRVHENEITKLRKSDFNGLNNVLVIELGGNPLKNSGIENGAFQGLKSLSYIRISDTNITAIPQGLPTSLTEVHLDGNKITKVDAPSLKGLINLSKLGLSFNSITVMENGSLANVPHLRELHLDNNKLLRVPAGLAQHKYIQVVYLHNNNISAVGQNDFCRAGHPSRKASYSAVSLYGNPVRYWEIFPNTFRCVYVRSAIQLGNYK

The MYXV of the present invention may include a LIGHT transgenic gene (MYXV-LIGHT). LIGHT (corresponding to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM)-a receptor expressed by T lymphocytes) is an immunomodulatory transduction that can be introduced into the MYXV genome Another example of reproductive genes. LIGHT is a member of the TNF superfamily that exhibits immunostimulatory activity. For example, LIGHT can promote T cell proliferation, Th1 chemotaxis and secretion of interleukins. The performance of LIGHT in the tumor microenvironment can promote the increase of the expression of chemokines, the increase of adhesion molecules and the infiltration of T cells. Due to its immunostimulatory activity and the extensive performance of its homologous receptor HveA, LIGHT is a promising candidate molecule for stimulating anti-tumor immune responses.

In some embodiments, the LIGHT transgenic gene comprises a sequence from a mammalian LIGHT gene. In some embodiments, the LIGHT transgenic gene comprises a sequence from the mouse LIGHT gene (mLIGHT). In some embodiments, the LIGHT transgenic gene comprises a sequence from the human LIGHT gene (huLIGHT). In some embodiments, the LIGHT transgenic gene encodes a secreted product. In some embodiments, the LIGHT transgenic gene encodes a product that is localized to the cell surface (eg, contains a transmembrane domain). As provided in Table 2, in some embodiments, LIGHT comprising a gene derived from SEQ ID NO: 13 - 15 in the sequence of any one of. Table 2 SEQ ID NO: description sequence 13 Human LIGHT/ TNFSF14, isoform 1 MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVRYGGVRGVRGSVVLPSGRLVGSVGLVGSVVLPSGLAVGSVGSVGSLVGSVGSLVGSVGSVGSLVGSVGLAFLRGLSYHDGALVVTKAGYYYIYSKR 14 Human/TNFSF14, isoform 2 MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELELLVSQQSAGEGRATSSSRVVLDERGALVGVVRSVLEGALVVVRSV 15 Mouse LIGHT/ TNFSF14 MESVVQPSVFVVDGQTDIPFRRLEQNHRRRRCGTVQVSLALVLLLGAGLATQGWFLLRLHQRLGDIVAHLPDGGKGSWEKLIQDQRSHQANPAAHLTGANASLIGIGGPLLWETRLGLAFLRGLTYHDGALVTMEPGYYYVYSKVQLSGVGCPKVYSKVQLSGVGCPKVVYSKVQLSGVGCPKVSPRGLRGLRGVRGFLG

The MYXV of the present invention may include a BiKE transgenic gene (MYXV-BiKE). In some embodiments, the BiKE transgenic gene contains sequences derived from one or more antibodies (e.g., one or more heavy chain variable domains, one or more light chain variable domains, one or more complementarity determining regions, CDR) or a combination thereof). In some embodiments, the BiKE transgenic gene contains sequences derived from one or more mammalian antibodies. In some embodiments, the BiKE transgene contains sequences derived from one or more mouse antibodies. In some embodiments, the BiKE transgenic gene comprises sequences derived from one or more humanized antibodies (huBiKE). In some embodiments, the BiKE transgene encodes a secreted product. In some embodiments, the BiKE transgenic gene encodes a product that is localized to the cell surface (eg, contains a transmembrane domain). As provided in Table 3, in some embodiments, from BIKE gene comprising SEQ ID NO: 16 - sequence of one or more of any of the 31. SEQ ID NO: 16 - 17 provide variable region sequences from an antibody having specificity for the CD138. SEQ ID NO: 18 - 19 provide variable region sequences from an antibody having specificity for the CD16. The identification is by the method of Kabat, SEQ ID NO: 20 - 25 provides CDR sequences from an antibody having specificity for the CD138. The identification is by the method of Kabat, SEQ ID NO: 26 - 31 provides CDR sequences from an antibody having specificity for the CD16. table 3 SEQ ID NO: description sequence 16 Anti-CD138 VH SQVQLQQSGSELMMPGASVKISCKATGYTFSNYWIEWVKQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSS 17 Anti-CD138 VL DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIK 18 Anti-CD16 VH QVTLKESGPGILQPSQTLSLTCSFSGFSLRTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSSNQVFLKIASVDTADTATYYCAQINPAWFAYWGQGTLVTVSA 19 Anti-CD16 VL DTVLTQSPASLAVSLGQRATISCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGIPARFSASGSGTDFTLNIHPVEEEDTATYYCQQSNEDPYTFGGGTKLEIK 20 Anti-CD138 HCDR1 NYWIE twenty one Anti-CD138 HCDR2 EILPGTGRTIYNEKFKG twenty two Anti-CD138 HCDR3 RDYYGNFYYAMDY twenty three Anti-CD138 LCDR1 SASQGINNYLN twenty four Anti-CD138 LCDR2 YTSTLQS 25 Anti-CD138 LCDR3 QQYSKLPRT 26 Anti-CD16 HCDR1 TSGMGVG 27 Anti-CD16 HCDR2 HIWWDDDKRYNPALKS 28 Anti-CD16 HCDR3 INPAWFAY 29 Anti-CD16 LCDR1 KASQSVDFDGDSFMN 30 Anti-CD16 LCDR2 TTSNLES 31 Anti-CD16 LCDR3 QQSNEDPYT

Bispecific natural killer and neutrophil conjugation molecule (BiKE) (CD138-CD16) is another example of an immunomodulatory transgene that can be introduced into the MYXV genome. BiKE (CD138-CD16) can guide natural killer (NK) cells and neutrophils to invade tumor targets, for example, by binding CD16 on the surface of NK cells and neutrophils, and CD138 on the surface of MM cells. This can cause NK/neutrophil activation, induce apoptosis of target cancer cells, and produce cytokines and chemokines in response to malignant targets (Gleason MK, Verneris MR, Todhunter DA, Zhang B, McCullar V, Zhou SX et al. Bispecific and trispecific killer cell engagers directly activate human NK cells through CD16 signaling and induce cytotoxicity and cytokine production. Mol Cancer Ther 2012;11(12):2674-84).

In some embodiments, disclosed herein is a recombinant MYXV construct that has one or more of these immunomodulatory transgenic genes to target hematological cancers, including MM. In the present invention, MYXV expressing the transgenic BiKE, Decorin or LIGHT exhibits selective infection and killing of primary human MM cells from patients with refractory diseases that are resistant to standard therapies. In addition, it was confirmed that these virus constructs can impair the survival rate of MM cells by inducing apoptosis and death of MM cells. It is worth noting that two types of MM cell killing can be observed: direct cytotoxicity to kill virus-infected MM cells, and "off target" to kill uninfected MM cells. Without wishing to be bound by theory, the killing of uninfected MM cells can be mediated by MYXV activated immune cells present in patient samples.

The sequence of the present invention may have at least 70% homology, at least 71% homology, at least 72% homology, at least 73% homology, at least 74% homology with the amino acid or nucleic acid sequence disclosed herein Homology, at least 75% homology, at least 76% homology, at least 77% homology, at least 78% homology, at least 79% homology, at least 80% homology, at least 81% homology , At least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, At least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology, at least 99.1% homology, at least 99.2% homology, at least 99.3% homology, at least 99.4 % Homology, at least 99.5% homology, at least 99.6% homology, at least 99.7% homology, at least 99.8% homology, at least 99.9% homology, at least 99.91% homology, at least 99.92% Homology, at least 99.93% homology, at least 99.94% homology, at least 99.95% homology, at least 99.96% homology, at least 99.97% homology, at least 99.98% homology, or at least 99.99% homology Source.

The transgenic gene of the present invention (for example, BiKE transgenic gene) can encode an antigen binding protein, such as one or more variable regions or complementarity determining regions (CDR) derived from antibodies. In some embodiments, the transgenic gene of the present invention (eg, BiKE transgenic gene) comprises one or more single chain variable fragments (scFv) derived from one or more antibodies. The scFv (single chain variable fragment) is a fusion protein that can include the VH and VL domains of an antibody linked by a peptide linker. For example, the BiKE transgene can contain two scFvs that allow the binding of two targets.

Antigen binding proteins can be engineered based on antibody variable regions or CDRs. The variable (V) region of an antibody mediates antigen binding and defines the specificity of a particular antibody to the antigen. Variable regions include relatively constant sequences called framework regions, and hypervariable regions with significantly different sequences between antibodies with different binding specificities. Inside the hypervariable region are amino acid residues that mainly determine the binding specificity of the antibody. The sequence containing these residues is called the complementarity determining region (CDR). One antigen binding site of an antibody contains six CDRs, three in the hypervariable region of the light chain and three in the hypervariable region of the heavy chain. The CDRs in the light chain are called L1, L2, and L3, and the CDRs in the heavy chain are called H1, H2, and H3. CDRs can also be named LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively. The effect of each CDR on antigen binding varies between antibodies. The length of the CDR can vary. For example, CDRs are often 5 to 14 residues in length, but CDRs are as short as 0 residues or as long as 25 residues or longer. Several methods can be used to predict or specify CDR sequences, such as Kabat, Chothia, IMGT, paratome, Martin, and AHo methods. These CDR prediction methods can use different numbering systems, for example because sequence insertions and deletions are numbered differently.

The antigen binding protein may comprise a part of an antibody, such as the antigen binding or variable region of a complete antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, scFv, mini-antibodies, diabodies, tri-functional antibodies, and tetra-functional antibodies And linear antibodies. Fab and Fab' are antigen-binding fragments, which may include the VH and CH1 domains of the heavy chain connected to the VL and CL domains of the light chain via disulfide bonds. F(ab')2 may include two Fab or Fab' connected by disulfide bonds. Fv can include VH and VL domains held together by non-covalent interactions. The scFv (single chain variable fragment) is a fusion protein that can include VH and VL domains connected by a peptide linker. The manipulation of the orientation of the VH and VL domains and the length of the linker can be used to form different forms of molecules, which can be monomers, dimers (bifunctional antibodies), trimers (trifunctional antibodies), or tetramers (tetrafunctional antibodies) .

In some embodiments, the transgenic gene of the present invention may encode a linker sequence (for example, a linker sequence between different domains of the protein encoded by the transgenic gene). In some embodiments, linkers are used to link antibody variable regions to form scFv. In some embodiments, linkers are used to join two scFvs to form BiKE. The length of the linker sequence can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.

The flexible linker may have a sequence of elongated segments containing glycine and serine residues. The smaller size of glycine and serine residues provides flexibility and allows mobility of the linked functional domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing the adverse interaction between the linker and the protein part. The flexible linker may also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and polar amino acids, such as lysine and glutamic acid, to improve solubility. The rigid linker may have, for example, an alpha helical structure. The alpha-helix rigid linker can act as a spacer between protein domains. The linker may include any one of the sequences in Table 4 or a repeated sequence thereof. SEQ ID NO: 32 - 37 provide a flexible linker. SEQ ID NO: 38 - 41 provide a rigid linker. Table 4 SEQ ID NO: sequence 32 GGGGS 33 GGGS 34 GG 35 KESGSVSSEQLAQFRSLD 36 EGKSSGSGSESKST 37 GSAGSAAGSGEF 38 EAAAK 39 EAAAR 40 PAPAP 41 AEAAAKEAAAKA

In some embodiments, the MYXV of the present invention may include one or more additional transgenic genes (for example, one or more transgenic genes other than one or more of Decorin, BiKE, and LIGHT).

In some embodiments, the MYXV of the present invention may include one or more non-immunomodulatory transgenic genes (for example, one or more non-immunomodulatory transgenic genes other than one or more of Decorin, BiKE, and LIGHT).

In some embodiments, the MYXV of the present invention may include one or more reporter transgenic genes (for example, one or more reporter transgenic genes other than one or more of Decorin, BiKE, and LIGHT). The reporter transgenic gene (or reporter gene) can be used to monitor or quantify MYXV in vitro, in vitro or in vivo. In some embodiments, the reported transgenic gene can be used to identify cells infected with MYXV of the present invention. For example, the MYXV of the present invention can express a fluorescent transgenic gene, and the infected cells can be identified by fluorescence (such as fluorescence microscopy or flow cytometry). In some embodiments, the reported transgenic genes can be used to quantify cells infected with MYXV of the present invention. For example, the MYXV of the present invention can express a fluorescent transgenic gene, and the infected cells can be quantified by fluorescence (for example, quantifying the number or proportion of infected cells by fluorescence microscopy or flow cytometry). In some embodiments, the reported transgenic gene can be used to quantify viral replication or viral load in cells infected with MYXV of the present invention. For example, the MYXV of the present invention can express a fluorescent transgenic gene, and the infected cells can be quantified by fluorescence (for example, the average fluorescence intensity of the cells is quantified by flow cytometry by fluorescence microscopy). In some embodiments, the MYXV of the present invention can express a reporter gene that can be used to quantify viral load or viral replication in vivo (for example, using an in vivo imaging system (IVIS) imaging).

The reported transgenic gene of the present invention can be expressed constitutively (for example, under the control of a constitutive promoter). The reported transgenic genes of the present invention can be expressed conditionally (for example, under the control of a conditional promoter, such as a promoter that is active or more active only in certain stages of the replication cycle).

Non-limiting examples of reporter genes include fluorescent proteins (e.g., green fluorescent protein (GFP), TdTomato, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), Verde Fluorescent protein (VFP), bright fluorescent protein (KFP), mCherry, mTangerine, mRaspberry, mPlum, DsRed, etc.) and enzymes and substrates involved in luminescence.

In some embodiments, MYXV of the present invention does not contain a reporter transgenic gene (for example, it does not encode any fluorescent or photoluminescent protein).

In addition to the expression of one or more immunomodulatory transgenic genes, the MYXV of the present invention can be modified to carry one or more other genes that can enhance the anti-cancer effect of MYXV therapy. Such genes can be genes involved in triggering cell apoptosis, or involved in the immune destruction of targeted infected cells, such as restoring cell reactivity to interferon, or causing cell surface markers (such as bacterial cell surface antigens) that stimulate antibody responses The gene of its performance. The MYXV of the present invention can be modified to express one or more genes involved in blocking neoplasia or cancer cell proliferation and growth, thereby preventing or reducing the division of cancer cells. In some embodiments, MYXV of the present invention can be modified to include therapeutic genes, such as genes involved in the synthesis of chemotherapeutic agents. In some embodiments, the MYXV of the present invention may include a transgenic gene that increases virus replication in cells of a specific species (for example, increases replication in human cancer cells for increasing the killing and suppression of human cancer cells). treatment method

In some embodiments, provided herein is a method of using the myxoma virus (MYXV) of the present invention to treat blood cancer in an individual. Hematological cancers may be hematological cancers including minimal residual disease (MRD) and/or drug-resistant MRD.

As disclosed in the following examples, in vitro studies have confirmed the ability of the MYXV construct of the present invention to significantly eliminate refractory primary multiple myeloma (MM) cells from patients whose standard therapies have failed. Research conducted with MYXV has shown that it can be a highly specific anticancer agent tropism for a variety of human and murine cancer types.

The treatment of the present invention (for example, treatment using MYXV-BiKE-Decorin, MYXV-LIGHT-Decorin or MYXV-Decorin) can include a variety of novel and advantageous aspects. For example, these virus constructs are far from the only oncolytic viruses described so far, which selectively target and directly eliminate drug-resistant primary human MM cells that have been directly infected by each virus (for example, expressing viral reporter genes (such as GFP ⁺ or TdTomato ⁺ ) CD138 ⁺ cells). In some embodiments, the MYXV of the present invention containing the transgenic gene can not only eliminate contaminated blood cancer cells by directly killing virus-infected cells, but also can kill uninfected cancer cells by enhancing "off target" (For example, immune cells activated by viruses). In some embodiments, the MYXV of the present invention containing the transgenic gene can cause enhanced killing of uninfected cancer cells compared to other viruses (such as unarmed viruses or viruses lacking transgenic genes). In some embodiments, MYXV of the present invention can exhibit enhanced "off-target" killing of uninfected MM cells (eg, CD138 ⁺ cells that are negative for viral reporter genes, such as GFP ^- or TdTomato ^- ). Without wishing to be bound by any particular theory, virus-enhanced killing of uninfected cells can be mediated by immune cells activated by MYXV (for example, immune cells activated by the transgenic gene, other viral components of the present invention, or a combination thereof) .

The use of MYXV-BiKE-Decorin, MYXV-LIGHT-Decorin or MYXV-Decorin to treat hematological malignancies (e.g., refractory and/or minimal residual disease (MRD) of hematological malignancies) may include advantages over current therapies (including chemical Therapies and stem cell transplantation) and other candidate oncolytic viruses. MYXV contains a finite tropism, which can, for example, allow viruses to infect human cancer cells, but not allow viruses to infect non-cancerous human cells. Unlike most viruses suitable for human pathogens, MYXV does not cause disease in humans, making it safe even for patients with compromised immune systems. The lack of pre-existing adaptive immunity against MYXV in the human population can be advantageous, for example, allowing viruses to infect and kill cancer cells without being quickly eliminated from them like viruses that have adapted to human pathogens.

In the ex vivo treatment method disclosed herein, MYXV is combined with cells (such as bone marrow (BM) cells and/or peripheral blood mononuclear cells (PBMC)) before reinfusing the cells back into cancer patients The incubation can be very fast, for example, it only needs to incubate the virus in vitro for 1 hour.

Therefore, aspects of the present invention provide a method of inhibiting and/or treating hematological cancers in individuals in need. In certain embodiments, the method includes administering to an individual, such as a human individual, MYXV of the present invention that exhibits one or more immunomodulatory transgenic genes, such as BiKE, LIGHT, and/or Decorin, thereby treating and/or inhibiting Hematological cancer of individuals in need. The individual may be a mammal. The individual may be a human.

In some embodiments, MYXV includes MYXV-LIGHT. In some embodiments, MYXV includes MYXV-Decorin. In some embodiments, MYXV comprises MYXV-BiKE. In some embodiments, MYXV includes LIGHT and Decorin. In some embodiments, MYXV includes LIGHT and BiKE. In some embodiments, MYXV includes Decorin and BiKE. In some embodiments, MYXV includes LIGHT, Decorin, and BiKE. LIGHT, Decorin, or BiKE may include sequences from humans, mice, mammals, or combinations thereof, and may include any of the sequences disclosed herein.

In some embodiments, the MYXV of the present invention includes a reporter transgenic gene (such as a fluorescent protein or a luminescent substrate or an enzyme). In some embodiments, the MYXV of the present invention includes one or more of LIGHT, Decorin, and BiKE, and further includes a reporter transgenic gene.

In some embodiments, MYXV of the present invention includes modification, insertion, deletion or destruction of one or more genes in the viral genome. For example, the MYXV of the present invention may include modification, insertion, deletion or destruction of any one or more of the following: M001R, M002R, M003.1R, M003.2R, M004.1R, M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2, M-T4, M-T5, M-T7 and SOD genes. In some embodiments, the deletion or destruction of the viral gene in MYXV of the present invention can reduce the disease caused by the virus in the host animal, regulate the tropism of the host cell, reduce the innate immune evasion of non-cancer cells, and regulate the immunity of infected cells. Signal transduction, regulating cell death pathways in infected cells, increasing the ability of virus replication in cancer cells or combinations thereof.

In some embodiments, the MYXV of the present invention includes one or more insertions, deletions, or substitutions in or near one or more genes related to host cell tropism (for example, rabbit cell tropism). In some embodiments, the one or more genes associated with rabbit cell tropism include M11L, M063, M135R, M136R, M-T2, M-T4, M-T5, M-T7, or a combination thereof. In some cases, the one or more genes associated with rabbit cell tropism include M135R, M136R, or a combination thereof.

In some embodiments, the MYXV of the present invention includes the modification, insertion, deletion or destruction of the M135R gene. In some embodiments, the MYXV of the present invention includes the deletion or destruction of the M135R gene. Deletion or destruction of the M135R gene can, for example, weaken the ability of MYXV of the present invention to cause disease in the host animal without compromising the ability of MYXV to exhibit anti-cancer effects (such as infecting and killing cancer cells, causing anti-tumor immune responses or a combination thereof) .

In some embodiments, MYXV includes MYXV-LIGHT, MYXV-BiKE, or MYXV-Decorin. In some embodiments, MYXV comprises MYXV-FLuc-LIGHT-TdTomato, MYXV-BiKE-GFP or MYXV-Decorin-GFP. In some embodiments, MYXV comprises MYXV-FLuc-huLIGHT-TdTomato, MYXV-huBiKE-GFP or MYXV-mDecorin-GFP. In some embodiments, MYXV includes MYXV-FLuc-huLIGHT, MYXV-huBiKE, or MYXV-mDecorin, that is, the fluorescent reporter gene is ignored.

MYXV can infect cells with defective innate antiviral responses (such as human cells). As used herein, having a "defective innate antiviral response" may refer to a cell that is unable to induce one or more antiviral defense mechanisms when exposed to a virus or when a virus invades. For example, the defective innate antiviral response may include failure to inhibit viral replication, failure to produce antiviral cytokines (such as interferon), and failure to respond to antiviral cytokines (such as inducing interferon response pathways) , Failure to induce cell apoptosis, failure to trigger recognition via innate immune receptors (such as pattern recognition receptors), or a combination thereof.

Defective innate antiviral response can be caused by various factors, such as malignant transformation, mutation, infection, genetic defect or environmental stress.

In some embodiments, the MYXV of the present invention is not administered to individuals who have a defective innate antiviral response caused by genetic defects, environmental stress, or infection (such as pre-existing infections of different pathogens).

In some embodiments, the MYXV of the present invention is administered to individuals who have a defective innate antiviral response caused by malignant transformation (such as cancer). Cells containing defective innate antiviral response may be cancer cells. Compared with normal cells, such as non-cancerous cells, cancer cells have weakened or insufficient innate antiviral response after exposure to or infection by viruses. This may include, for example, cancer cells that do not respond to interferons (such as type I interferons), and/or cancer cells that have reduced or insufficient apoptotic response or induction of the apoptotic pathway. In some embodiments of the method, the MYXV of the present invention can infect cells with defective innate antiviral response. In some embodiments, the cell is a mammalian cancer cell. In some embodiments, the cells are human cancer cells, such as human blood cancer cells.

In some embodiments, MYXY of the present invention is used to treat cancer. It is inferred that the examples for multiple myeloma provided herein are applicable to other blood cancers. The types of cancers that can be treated according to the disclosed methods include (but are not limited to) hematological cancers, such as leukemia, lymphoma, and myeloma, such as multiple myeloma (MM); active multiple myeloma; smoldering multiple Myeloma; Plasmacytoma; Solitary osteoplasmacytoma; Extramedullary plasmacytoma; Light chain myeloma; Nonsecretory myeloma; Immunoglobulin G (IgG) myeloma; Immunoglobulin A (IgA) myeloma ; Immunoglobulin M (IgM) myeloma; Immunoglobulin D (IgD) myeloma; Immunoglobulin E (IgE) myeloma; Hyperdiploid multiple myeloma; Non-hyperdiploid multiple myeloma; Hodgkin's lymphoma (Hodgkin lymphoma); non-Hodgkin's lymphoma; acute lymphoblastic leukemia; acute myelogenous leukemia; essential thrombocythemia; polycythemia vera; primary myelofibrosis; systemic Chronic mastocytosis; chronic myelogenous leukemia; chronic neutrophil leukemia; chronic eosinophilic leukemia; refractory anemia with ring-shaped iron embryonic blood cells; refractory cytopenia with multi-lineage dysplasia; with excess Refractory anemia with blast type 1; Refractory anemia with excess blast type 2; Myelodysplastic syndrome (MDS) with isolated del (5q); Unclassified MDS; Chronic myelomonocytic leukemia (CML) ); atypical chronic myelogenous leukemia; juvenile myelomonocytic leukemia; myeloproliferative/myelodysplastic syndrome-not classified; B lymphoblastic leukemia/lymphoma; T lymphoblastic leukemia/lymphoma; diffuse Large B-cell lymphoma; Primary central nervous system lymphoma; Primary mediastinal B-cell lymphoma; Burkitt lymphoma/leukemia; Follicular lymphoma; Chronic lymphocytic leukemia (CLL)/ Small lymphocytic lymphoma; B-cell prolymphocytic leukemia; lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia; mantle cell lymphoma; marginal zone lymphoma; lymphocytes after transplantation Proliferative disorders; HIV-related lymphoma; primary exudative lymphoma; intravascular large B-cell lymphoma; primary skin primary cutaneous B-cell lymphoma; hairy cell leukemia; and gamma of unknown significance Globulinism; pleomorphic large cell lymphoma, angioimmunoblastic T-cell lymphoma, hepatocellular splenic T-cell lymphoma, B-cell lymphoma, reticuloendothelial hyperplasia, reticulocytosis, mucosal-related Lymphoid tissue lymphoma, B-cell chronic lymphocytic leukemia, Waldenstrom's macroglobulinemia, lymphoma-like granuloma, Hodgkin's lymphoma with prominent nodular lymphocytes, plasma cell leukemia, acute hypererythrocytes Diseases and erythroleukemia, acute red blood disease (erythremic myelosis), acute red blood cell leukemia, Heilmeyer-Schöner disease (Heilmeyer-Schöner d isease), acute megakaryoblastic leukemia, mast cell leukemia, panmyelopathy, acute panmyelopathy with myelofibrosis, lymphosarcoma cell leukemia, stem cell leukemia, chronic leukemia of unspecified cell type, of unspecified cell type Subacute leukemia, accelerated chronic myelogenous leukemia, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute bone marrow dendritic cells Leukemia, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, B-cell chronic lymphocytic leukemia, B-cell leukemia, chronic myelogenous leukemia, chronic idiopathic myelofibrosis, Kahler's disease , Myeloma disease, solitary myeloma, plasma cell leukemia, vascular central immune proliferative disease, lymphoid granuloma, angioimmunoblastic lymphadenopathy, T-γ lymphoproliferative disease, Waldenstrom's giant bulb Proteinemia, alpha heavy chain disease, gamma heavy chain disease and Franklin's disease. In some embodiments, the blood cancer is multiple myeloma. In some embodiments, the cancer is a blood cancer. In certain embodiments, the cancer comprises multiple myeloma.

In some embodiments, provided herein are methods of treating blood cancers (eg, inhibit, alleviate, stabilize, reduce or delay the progression of blood cancers). In some embodiments, the methods include administering MYXV of the present invention to individuals in need to treat blood cancers. In some embodiments, the method further includes selecting individuals who have or are suspected of having blood cancers, such as human individuals.

The MYXV of the present invention can be administered in an amount effective to treat blood cancer. The amount may be sufficient to reduce the number of cancer cells in the individual (e.g., the concentration of cancer cells in the blood of the individual).

The effective amount to be administered to the individual may vary depending on many factors, such as the pharmacodynamic properties of MYXV, the mode of administration, the age, health and weight of the individual, the nature and degree of the disease, frequency of treatment, and concurrent treatment (if any) The type of virus and the toxicity and potency of the virus.

Depending on the individual’s clinical response, MYXV is initially administered in a suitable amount, which can be adjusted as needed. The effective amount of virus can be determined empirically and depends on the maximum amount of MYXV that can be safely administered and the minimum amount of virus that produces the desired result.

In order to produce the same clinical effect when the virus is administered systemically as the clinical effect achieved by injecting the virus into the disease site, it may be necessary to administer a significantly higher amount of the virus. However, the appropriate dose level should be the smallest amount that will achieve the desired result.

The concentration of the virus to be administered will vary depending on the toxicity of the specific MYXV strain to be administered and the nature of the targeted cells. In one embodiment, a dose of less than about 3×10 ¹⁰ focus forming units (“ffu”) (also referred to as “infectious units”) is administered to a human individual. In various embodiments, it can be administered And about 10 ² to about 10 ⁹ pfu, about 10 ² to about 10 ⁷ pfu, about 10 ³ to about 10 ⁶ pfu, or about 10 ⁴ to about 10 ⁵ pfu in a single dose.

In some embodiments, a certain dose of the focal forming unit (FFU) or plaque forming unit (PFU) of MYXV of the present invention is administered to the individual.

In some embodiments, the dose of MYXV administered to the individual is at least 1×10^2, 2×10^2, 3×10^2, 4×10^2, 5×10^2, 6×10^ 2. 7×10^2, 8×10^2, 9×10^2, 1×10^3, 2×10^3, 3×10^3, 4×10^3, 5×10^3, 6×10^3, 7×10^3, 8×10^3, 9×10^3, 1×10^4, 2×10^4, 3×10^4, 4×10^4, 5× 10^4, 6×10^4, 7×10^4, 8×10^4, 9×10^4, 1×10^5, 2×10^5, 3×10^5, 4×10^ 5, 5×10^5, 6×10^5, 7×10^5, 8×10^5, 9×10^5, 1×10^6, 2×10^6, 3×10^6, 4×10^6, 5×10^6, 6×10^6, 7×10^6, 8×10^6, 9×10^6, 1×10^7, 2×10^7, 3× 10^7, 4×10^7, 5×10^7, 6×10^7, 7×10^7, 8×10^7, 9×10^7, 1×10^8, 2×10^ 8, 3×10^8, 4×10^8, 5×10^8, 6×10^8, 7×10^8, 8×10^8, 9×10^8, 1×10^9, 2×10^9, 3×10^9, 4×10^9, 5×10^9, 6×10^9, 7×10^9, 8×10^9, 9×10^9, 1× 10^10, 2×10^10, 3×10^10, 4×10^10, 5×10^10, 6×10^10, 7×10^10, 8×10^10, 9×10^ 10, 1×10^11, 2×10^11, 3×10^11, 4×10^11, 5×10^11, 6×10^11, 7×10^11, 8×10^11, 9×10^11, 1×10^12, 2×10^12, 3×10^12, 4×10^12, 5×10^12, 6×10^12, 7×10^12, 8× 10^12, 9×10^12, 1×10^13, 2×10^13, 3×10^13, 4×10^13, 5×10^13, 6×10^13, 7×10^ 13, 8×10^13, 9×10^13, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6×10^14, 7×10^14, 8×10^14, 9×10^14, 1×10^15, 2×10^15, 3×10^15, 4×10^15, 5×10^15, 6× 10^15, 7×10^15, 8×10^15, 9×10^15, 1×10^16, 2×10^16, 3×10^16, 4×10^16, 5×10^ 16. 6×10^16, 7×10^16, 8×10^16, 9×10^16, 1×10^17, 2×10^17, 3×10^17, 4×10^17, 5× 10^17, 6×10^17, 7×10^17, 8×10^17, 9×10^17, 1×10^18, 2×10^18, 3×10^18, 4×10^ 18, 5×10^18, 6×10^18, 7×10^18, 8×10^18, 9×10^18, 1×10^19, 2×10^19, 3×10^19, 4×10^19, 5×10^19, 6×10^19, 7×10^19, 8×10^19, 9×10^19, 1×10^20, 2×10^20, 3× 10^20, 4×10^20, 5×10^20, 6×10^20, 7×10^20, 8×10^20 or 9×10^20 FFU or PFU MYXV of the present invention.

In some embodiments, the dose of MYXV administered to the individual is at most 1×10^2, 2×10^2, 3×10^2, 4×10^2, 5×10^2, 6×10^ 2. 7×10^2, 8×10^2, 9×10^2, 1×10^3, 2×10^3, 3×10^3, 4×10^3, 5×10^3, 6×10^3, 7×10^3, 8×10^3, 9×10^3, 1×10^4, 2×10^4, 3×10^4, 4×10^4, 5× 10^4, 6×10^4, 7×10^4, 8×10^4, 9×10^4, 1×10^5, 2×10^5, 3×10^5, 4×10^ 5, 5×10^5, 6×10^5, 7×10^5, 8×10^5, 9×10^5, 1×10^6, 2×10^6, 3×10^6, 4×10^6, 5×10^6, 6×10^6, 7×10^6, 8×10^6, 9×10^6, 1×10^7, 2×10^7, 3× 10^7, 4×10^7, 5×10^7, 6×10^7, 7×10^7, 8×10^7, 9×10^7, 1×10^8, 2×10^ 8, 3×10^8, 4×10^8, 5×10^8, 6×10^8, 7×10^8, 8×10^8, 9×10^8, 1×10^9, 2×10^9, 3×10^9, 4×10^9, 5×10^9, 6×10^9, 7×10^9, 8×10^9, 9×10^9, 1× 10^10, 2×10^10, 3×10^10, 4×10^10, 5×10^10, 6×10^10, 7×10^10, 8×10^10, 9×10^ 10, 1×10^11, 2×10^11, 3×10^11, 4×10^11, 5×10^11, 6×10^11, 7×10^11, 8×10^11, 9×10^11, 1×10^12, 2×10^12, 3×10^12, 4×10^12, 5×10^12, 6×10^12, 7×10^12, 8× 10^12, 9×10^12, 1×10^13, 2×10^13, 3×10^13, 4×10^13, 5×10^13, 6×10^13, 7×10^ 13, 8×10^13, 9×10^13, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6×10^14, 7×10^14, 8×10^14, 9×10^14, 1×10^15, 2×10^15, 3×10^15, 4×10^15, 5×10^15, 6× 10^15, 7×10^15, 8×10^15, 9×10^15, 1×10^16, 2×10^16, 3×10^16, 4×10^16, 5×10^ 16. 6×10^16, 7×10^16, 8×10^16, 9×10^16, 1×10^17, 2×10^17, 3×10^17, 4×10^17, 5× 10^17, 6×10^17, 7×10^17, 8×10^17, 9×10^17, 1×10^18, 2×10^18, 3×10^18, 4×10^ 18, 5×10^18, 6×10^18, 7×10^18, 8×10^18, 9×10^18, 1×10^19, 2×10^19, 3×10^19, 4×10^19, 5×10^19, 6×10^19, 7×10^19, 8×10^19, 9×10^19, 1×10^20, 2×10^20, 3× 10^20, 4×10^20, 5×10^20, 6×10^20, 7×10^20, 8×10^20 or 9×10^20 FFU or PFU MYXV of the present invention.

In some embodiments, a certain dose per kilogram of body weight of the lesion forming unit (FFU) or plaque forming unit (PFU) of MYXV of the present invention is administered to the individual.

In some embodiments, the dose of MYXV administered to the individual is at least 1×10^2, 2×10^2, 3×10^2, 4×10^2, 5×10^2 per kilogram of the body weight of the individual. 6×10^2, 7×10^2, 8×10^2, 9×10^2, 1×10^3, 2×10^3, 3×10^3, 4×10^3, 5× 10^3, 6×10^3, 7×10^3, 8×10^3, 9×10^3, 1×10^4, 2×10^4, 3×10^4, 4×10^ 4, 5×10^4, 6×10^4, 7×10^4, 8×10^4, 9×10^4, 1×10^5, 2×10^5, 3×10^5, 4×10^5, 5×10^5, 6×10^5, 7×10^5, 8×10^5, 9×10^5, 1×10^6, 2×10^6, 3× 10^6, 4×10^6, 5×10^6, 6×10^6, 7×10^6, 8×10^6, 9×10^6, 1×10^7, 2×10^ 7, 3×10^7, 4×10^7, 5×10^7, 6×10^7, 7×10^7, 8×10^7, 9×10^7, 1×10^8, 2×10^8, 3×10^8, 4×10^8, 5×10^8, 6×10^8, 7×10^8, 8×10^8, 9×10^8, 1× 10^9, 2×10^9, 3×10^9, 4×10^9, 5×10^9, 6×10^9, 7×10^9, 8×10^9, 9×10^ 9, 1×10^10, 2×10^10, 3×10^10, 4×10^10, 5×10^10, 6×10^10, 7×10^10, 8×10^10, 9×10^10, 1×10^11, 2×10^11, 3×10^11, 4×10^11, 5×10^11, 6×10^11, 7×10^11, 8× 10^11, 9×10^11, 1×10^12, 2×10^12, 3×10^12, 4×10^12, 5×10^12, 6×10^12, 7×10^ 12, 8×10^12, 9×10^12, 1×10^13, 2×10^13, 3×10^13, 4×10^13, 5×10^13, 6×10^13, 7×10^13, 8×10^13, 9×10^13, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6× 10^14, 7×10^14, 8×10^14, 9×10^14, 1×10^15, 2×10^15, 3×10^15, 4×10^15, 5×10^ 15, 6×10^15, 7×10^15, 8×10^15, 9×10^15, 1×10^16, 2×10^16, 3×10^16, 4×10^16, 5 ×10^16, 6×10^16, 7×10^16, 8×10^16, 9×10^16, 1×10^17, 2×10^17, 3×10^17, 4×10 ^17, 5×10^17, 6×10^17, 7×10^17, 8×10^17, 9×10^17, 1×10^18, 2×10^18, 3×10^18 , 4×10^18, 5×10^18, 6×10^18, 7×10^18, 8×10^18, 9×10^18, 1×10^19, 2×10^19, 3 ×10^19, 4×10^19, 5×10^19, 6×10^19, 7×10^19, 8×10^19, 9×10^19, 1×10^20, 2×10 ^20, 3×10^20, 4×10^20, 5×10^20, 6×10^20, 7×10^20, 8×10^20 or 9×10^20 FFU or PFU of the present invention Of MYXV.

In some embodiments, the dose of MYXV administered to the individual is at most 1×10^2, 2×10^2, 3×10^2, 4×10^2, 5×10^2, per kilogram of the individual's body weight. 6×10^2, 7×10^2, 8×10^2, 9×10^2, 1×10^3, 2×10^3, 3×10^3, 4×10^3, 5× 10^3, 6×10^3, 7×10^3, 8×10^3, 9×10^3, 1×10^4, 2×10^4, 3×10^4, 4×10^ 4, 5×10^4, 6×10^4, 7×10^4, 8×10^4, 9×10^4, 1×10^5, 2×10^5, 3×10^5, 4×10^5, 5×10^5, 6×10^5, 7×10^5, 8×10^5, 9×10^5, 1×10^6, 2×10^6, 3× 10^6, 4×10^6, 5×10^6, 6×10^6, 7×10^6, 8×10^6, 9×10^6, 1×10^7, 2×10^ 7, 3×10^7, 4×10^7, 5×10^7, 6×10^7, 7×10^7, 8×10^7, 9×10^7, 1×10^8, 2×10^8, 3×10^8, 4×10^8, 5×10^8, 6×10^8, 7×10^8, 8×10^8, 9×10^8, 1× 10^9, 2×10^9, 3×10^9, 4×10^9, 5×10^9, 6×10^9, 7×10^9, 8×10^9, 9×10^ 9, 1×10^10, 2×10^10, 3×10^10, 4×10^10, 5×10^10, 6×10^10, 7×10^10, 8×10^10, 9×10^10, 1×10^11, 2×10^11, 3×10^11, 4×10^11, 5×10^11, 6×10^11, 7×10^11, 8× 10^11, 9×10^11, 1×10^12, 2×10^12, 3×10^12, 4×10^12, 5×10^12, 6×10^12, 7×10^ 12, 8×10^12, 9×10^12, 1×10^13, 2×10^13, 3×10^13, 4×10^13, 5×10^13, 6×10^13, 7×10^13, 8×10^13, 9×10^13, 1×10^14, 2×10^14, 3×10^14, 4×10^14, 5×10^14, 6× 10^14, 7×10^14, 8×10^14, 9×10^14, 1×10^15, 2×10^15, 3×10^15, 4×10^15, 5×10^ 15, 6×10^15, 7×10^15, 8×10^15, 9×10^15, 1×10^16, 2×10^16, 3×10^16, 4×10^16, 5 ×10^16, 6×10^16, 7×10^16, 8×10^16, 9×10^16, 1×10^17, 2×10^17, 3×10^17, 4×10 ^17, 5×10^17, 6×10^17, 7×10^17, 8×10^17, 9×10^17, 1×10^18, 2×10^18, 3×10^18 , 4×10^18, 5×10^18, 6×10^18, 7×10^18, 8×10^18, 9×10^18, 1×10^19, 2×10^19, 3 ×10^19, 4×10^19, 5×10^19, 6×10^19, 7×10^19, 8×10^19, 9×10^19, 1×10^20, 2×10 ^20, 3×10^20, 4×10^20, 5×10^20, 6×10^20, 7×10^20, 8×10^20 or 9×10^20 FFU or PFU of the present invention Of MYXV.

The MYXV of the present invention can be administered at any desired time interval. In some embodiments, MYXV may be administered hourly. In some embodiments, MYXV may be about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, Administer at 26, 28, 30, 32, 36, 40, 44, or 48 hours. In some embodiments, MYXV may be twice a day, once a day, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, every Once a month, once every five weeks, once every six weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every four months, once every six months, once every six Invest once a month.

The MYXV of the present invention can be administered to an individual in a therapeutically effective amount in various forms and routes, including, for example, systemic, oral, topical, parenteral, intravenous injection, intravenous infusion, intratumoral injection, subcutaneous injection, intramuscular injection. Intradermal injection, intradermal injection, intraperitoneal injection, intracerebral injection, subarachnoid injection, intraspinal injection, intrasternal injection, intraarticular injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, Intra-arterial administration, intrathecal administration, inhalation, intralesional administration, intradermal administration, epidural administration, absorption through epithelial or mucosal skin (such as oral mucosa, rectum and intestinal mucosa), intrasaccular administration And, subcapsular administration, intracardiac administration, transtracheal administration, subcutaneous administration, subarachnoid administration, subcapsular administration, intraspinal administration, or intrasternal administration.

In some embodiments, the virus is administered systemically. In some embodiments, the virus is administered at the disease site by injection. In some embodiments, the virus is administered orally. In some embodiments, the virus is administered parenterally.

The MYXV of the present invention (for example, expressing one or more immunomodulatory transgenic genes, such as BiKE, LIGHT, and/or Decorin) can be administered as the sole therapy or can be administered in combination with one or more other therapies. In some embodiments, the MYXV of the present invention is administered in combination with chemotherapy, immunotherapy, cell therapy, radiation therapy, stem cell transplantation (such as autologous stem cell transplantation), or a combination thereof. For example, MYXV that exhibits one or more immunomodulatory transgenes, such as BiKE, LIGHT and/or Decorin, can be administered before or after another treatment, such as administration of radiotherapy or conventional chemotherapy drugs and/or Stem cell transplantation, such as autologous stem cell transplantation or allogeneic stem cell transplantation (eg, HLA-matched, HLA-mismatched and/or haploid transplantation).

In some embodiments, MYXV of the present invention can be combined with immune checkpoint modulators. Immune checkpoint modulators include (but are not limited to) PD-L1 inhibitors, such as durvalumab (Imfinzi) from AstraZeneca, atezolizumab (MPDL3280A) from Genentech, EMD Avilumab from Serono/Pfizer, CX-072 from CytomX Therapeutics, FAZ053 from Novartis Pharmaceuticals, KN035 from 3D Medicine/Alphamab, LY3300054 from Eli Lilly or M7824 from EMD Serono (anti-PD- L1/TGFβ trap); PD-L2 inhibitors, such as AMP-224 (Amplimmune) and rHIgM12B7 from GlaxoSmithKline; PD-1 inhibitors, such as nivolumab (Opdivo) from Bristol-Myers Squibb, pembrolizumab (Keytruda) from Merck, AGEN 2034 from Agenus, BGB-A317 from BeiGene, Bl-754091 from Boehringer-Ingelheim Pharmaceuticals, CBT-501 (genolimzumab) from CBT Pharmaceuticals, INCSHR1210 from Incyte, JNJ-63723283 from Janssen Research & Development, MedImmune’s MEDI0680, MacroGenics’ MGA 012, Novartis Pharmaceuticals’ PDR001, Pfizer’s PF-06801591, Regeneron Pharmaceuticals/Sanofi’s REGN2810 (SAR439684), or TESARO’s TSR-042; CTLA-4 inhibitors such as Bristol Meyers Squibb’s ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), tremelimumab from Pfizer (CP-675,206, ticilimumab) or AGEN 1884 from Agenus; LAG3 inhibitors, such as from Bristol- Myers Squib b BMS-986016, IMP701 from Novartis Pharmaceuticals, LAG525 from Novartis Pharmaceuticals, or REGN3767 from Regeneron Pharmaceuticals; B7-H3 inhibitors, such as enblotuzumab (MGA271) from MacroGenics; KIR inhibition Agents, such as Lirilumab (IPH2101; BMS-986015) from Innate Pharma; CD137 inhibitors, such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 ( Anti-4-1BB, PF-2566, Pfizer) or XmAb-5592 (Xencor); and PS inhibitors such as Bavituximab (Bavituximab). In some embodiments, MYXV is combined with antibodies or antigen-binding fragments thereof, RNAi molecules or small molecules, which act on or have specificity for, for example, TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR , ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2 or SLAM.

The MYXV of the present invention can be prepared using standard techniques. For example, the virus can be prepared by infecting cultured rabbit cells or immortalizing human or primate cells with the MYXV strain to be used, so that the infection progresses so that the virus replicates in the cultured cells, and It can be released by standard methods used to destroy the cell surface and thereby release virus particles for collection. Once collected, the virus titer can be determined, for example, by infecting a fusion lawn of rabbit cells and performing a plaque assay (see Mossman et al. (1996) Virology 215:17-30, which is incorporated herein by reference in its entirety) . Cell Delivery of MYXV

In some embodiments, a novel delivery strategy is further disclosed herein, in which the MYXV of the present invention is first adsorbed to cells, and the cells are administered to the individual. This method can deliver the MYXV of the present invention to the disease site via virus-carrying "vector" cells. In some embodiments, this cell-assisted virus delivery method can reduce or eliminate tumor burden and increase the survival rate of individuals.

Delivery of MYXV via carrier cells represents a new potential treatment option for hematological cancers. In some embodiments, the MYXV of the present invention is adsorbed to white blood cells (for example, white blood cells of bone marrow and/or peripheral blood), and the white blood cells are infused into the individual. Preloading white blood cells with MYXV ex vivo can be used for multiple myeloma (MM) and for any other blood cancers disclosed herein before the white blood cells are infused into cancer-carrying recipients. In some embodiments, preloading leukocytes with MYXV ex vivo before leukocyte transfusion into cancer-carrying recipients can effectively treat any cancer that undergoes leukocyte localization and infiltration to remote tumor sites.

In some embodiments, the combined "white blood cell/MYXV" therapy causes increased cancer cell death in the tumor bed to enhance anti-tumor immunogenicity. For example, in some embodiments, MYXV of the present invention (for example, MYXV that exhibits one or more immunomodulatory transgenes, such as BiKE, LIGHT, and/or Decorin) is pre-adsorbed or pre-infected with leukocytes Migration and delivery to cancer sites, such as bone marrow beds with minimal residual disease (MRD). This systemic delivery method is sometimes referred to as "ex vivo viral therapy" or EVV (also known as EV2) because the virus is first delivered to white blood cells before infusion into the patient.

In some embodiments, the cell-mediated delivery of MYXV increases the level of direct killing of infected blood cancer cells and (though not bound by theory) acts as an activator of the host immune system, which can cause long-term cancer regression. This can provide a new method for the treatment of hematological cancers in the bone and/or lymph nodes, which has proven to be difficult to treat under current treatment conditions.

Therefore, in certain embodiments, the method of the present invention comprises administering white blood cells to an individual suffering from cancer, the white blood cells comprising adsorbed MYXV of the present invention (for example, one or more immunomodulatory transgenic genes, such as BiKE). , LIGHT and/or Decorin's MYXV), thereby treating and/or inhibiting cancer in an individual.

In some embodiments, the MYXV of the present invention is adsorbed to white blood cells (for example, white blood cells of bone marrow and/or peripheral blood), and the white blood cells are infused into the individual. White blood cells can be derived from bone marrow (e.g., from bone marrow aspirates or bone marrow slices). White blood cells can be derived from blood (e.g., peripheral blood mononuclear cells). In some embodiments, the white blood cells are obtained from an individual, such as an individual who has cancer, is adsorbed by MYXV and re-infused into the individual (e.g., as an autologous cell transplant). In some embodiments, the white blood cells are obtained from one or more allogeneic donors (eg, HLA-matched, HLA-mismatched, or haploid donors). In some embodiments, the white blood cells are obtained from HLA-matched sibs.

Before or after the adsorption of MYXV of the present invention, white blood cells can be sorted or purified by, for example, the following: red blood cell lysis, density gradient centrifugation (e.g. Ficol-Paque), leukocyte depletion, techniques including antibodies or their derivatives (e.g. Positive or negative selection via fluorescence activated cell sorting or magnetic activated cell sorting) or any combination thereof. In some embodiments, the white blood cells are sorted or purified to enrich cancer cells before or after the adsorption of MYXV of the present invention (for example, cells exhibiting cancer-related markers, such as CD138 for multiple myeloma cells). In some embodiments, the white blood cells are sorted or purified before or after the MYXV of the present invention is adsorbed to enrich non-cancer cells. In some embodiments, the cells are sorted or purified before or after the MYXV of the present invention is adsorbed to enrich one or more cell subpopulation cells (e.g., monocytes, lymphocytes, B cells, plasma cells, T cells, Neutrophils, basophils, eosinophils, megakaryocytes, NK cells, NKT cells, mast cells, innate lymphocytes, common bone marrow precursors, common lymphoid precursors, bone marrow blasts, monocytes, lymph Mother cells, prolymphocytes, blood blast cells, megakaryocytes, promegakaryocytes, stem cells, pre-B cells, precursors thereof, or any combination thereof. In some embodiments, the MYXV of the present invention is adsorbed to white blood cells, and the white blood cells are rich The set includes MYXV (eg, cells with MYXV binding and/or internalization).

Leukocytes can be stored (eg cryopreservation) before or after the MYXV of the present invention is adsorbed. In some embodiments, white blood cells can be cryopreserved, and then thawed, and then infused into the individual.

In some embodiments, the method includes adsorbing the MYXV of the present invention to the surface of white blood cells (eg, peripheral blood mononuclear cells, bone marrow cells, or purified/enriched subpopulations thereof). In some embodiments, adsorbing myxoma virus onto the surface of white blood cells includes exposing the white blood cells to MYXV under specific conditions that permit MYXV to bind to the surface of mononuclear peripheral blood cells and/or bone marrow cells. In some embodiments, the method includes infecting white blood cells with MYXV of the present invention. In some embodiments, infecting white blood cells with MYXV of the present invention involves exposing white blood cells to MYXV under specific conditions that permit internalization of MYXV into white blood cells. Exposure of white blood cells to MYXV may include any suitable reagents or conditions (for example, sterile cell culture medium, medium supplements, and appropriate incubation conditions that allow absorption and/or infection of white blood cells and maintain the survival rate of white blood cells).

MYXV and white blood cells can be exposed to each other at any ratio that allows viruses to adsorb to white blood cells. In some embodiments, adsorbing myxoma virus onto the surface of white blood cells includes exposing white blood cells to MYXV at the following multiplicity of infection (MOI): each white blood cell is about 0.000001, 0.00001, 0.0001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1×10^4, 1×10^5, 1×10^6, 1×10^9, 1×10^10, 1×10^11, 1×10^12, 1×10^13, 1×10^14 or 1×10^15 viruses.

In some embodiments, adsorbing myxoma virus onto the surface of white blood cells comprises exposing the white blood cells to MYXV at the following magnification of infection (MOI): each white blood cell is at least 0.000001, 0.00001, 0.0001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1× 10^4, 1×10^5, 1×10^6, 1×10^9, 1×10^10, 1×10^11, 1×10^12, 1×10^13, 1×10^ 14 or 1×10^15 viruses.

In some embodiments, adsorbing myxoma virus onto the surface of white blood cells includes exposing white blood cells to MYXV at the following magnification of infection (MOI): up to 0.000001, 0.00001, 0.0001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1× 10^4, 1×10^5, 1×10^6, 1×10^9, 1×10^10, 1×10^11, 1×10^12, 1×10^13, 1×10^ 14 or 1×10^15 viruses.

In some embodiments, adsorbing myxoma virus onto the surface of white blood cells includes exposing the white blood cells to MYXV at a magnification of infection (MOI) between the following values, for example, about 0.000001 to 1×10^15 per white blood cell, 0.0001 to 1×10^6, 0.001 to 1×10^4, 0.001 to 1000, 0.001 to 100, 0.001 to 10, 0.001 to 1, 0.001 to 0.1, 0.001 to 0.01, 0.01 to 1×10^4, 0.01 to 1000, 0.01 to 100, 0.01 to 10, 0.01 to 1, 0.01 to 0.1, 0.1 to 1×10^4, 0.1 to 1000, 0.1 to 100, 0.1 to 10, 0.1 to 1, 1 to 1×10^4, 1 to 1000, 1 to 100, or 1 to 10 viruses.

In some embodiments, adsorbing myxoma virus onto the surface of white blood cells includes exposing the white blood cells to MYXV at a magnification of infection (MOI) between about 0.1 and 10. In some embodiments, adsorbing the myxoma virus onto the surface of the white blood cell includes exposing the white blood cell to MYXV at a magnification of infection (MOI) between about 0.01 and 100. In some embodiments, adsorbing the myxoma virus onto the surface of the white blood cell includes exposing the white blood cell to MYXV at a magnification of infection (MOI) between about 0.001 and 1000.

In some embodiments, the white blood cells are in contact with or adsorbed by the MYXV of the present invention for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes. Minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 20 hours, 22 hours Or a 24-hour period.

In some embodiments, the white blood cells are in contact with or adsorbed by the MYXV of the present invention for at least the following period of time: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 2.5 hours, 3 hours, 3.5 hours , 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 20 Hours, 22 hours, 24 hours or longer.

In some embodiments, the leukocytes are in contact with or adsorbed to the MYXV of the present invention for a period of at most the following: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 2.5 hours, 3 hours, 3.5 hours , 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 18 hours, 20 Hours, 22 hours, 24 hours or less.

In some embodiments, the BM or PBMC cells are contacted or adsorbed with the MYXV construct ex vivo for about one hour. Other in vitro methods

As disclosed herein, MYXV can selectively infect cells with defective innate antiviral responses and can be used as an indicator of such cell defects. Therefore, cells removed from an individual can be analyzed for defects in innate antiviral response using the method of the present invention. Such determinations may indicate that when combined with other indicators, the individual may suffer from a particular disease condition, such as cancer. Cells can be removed from individuals (including human individuals) using known biopsy methods. The biopsy method will depend on the location and type of cells to be tested. The cells can be cultured and exposed to MYXV, for example, by adding live MYXV to the culture medium. The magnification of infection (MOI) can be changed using positive control cell cultures known to be infected when exposed to MYXV to determine the best MOI for a given cell type, density and culture technique.

The amount of MYXV added to the cultured cells may vary depending on the cell type, culture method, and virus strain.

The infectivity of cultured cells with MYXV can be determined by various methods known to those skilled in the art, including the ability of MYXV to cause cell death. It can also involve adding reagents to the cell culture to complete an enzymatic or chemical reaction with the viral expression product. The viral expression product can be inserted into the MYXV genome to report gene expression.

In one embodiment, MYXV can be modified to improve the ease of detecting infection status. For example, MYXV can be genetically modified to represent a marker that can be easily detected by phase contrast microscopy, fluorescence microscopy, or by radiography. The label may be a fluorescent protein or an enzyme that can participate in colorimetric or radiolabeling reactions. In some embodiments, the marker may be a gene product that interrupts or inhibits the specific function of the cell under test. Pharmaceutical composition

The MYXV of the present invention or the cells containing the MYXV of the present invention can be formulated as an ingredient in a pharmaceutical composition. Therefore, in some embodiments, the present invention provides a pharmaceutical composition comprising a myxoma virus expressing one or more immunomodulatory transgenic genes (such as BiKE, LIGHT, and/or Decorin), and a pharmaceutically acceptable dilution Agents or excipients. The composition may contain pharmaceutically acceptable concentrations of salt, buffers, preservatives and various compatible carriers.

The pharmaceutical composition may contain additional therapeutic agents, such as additional anticancer agents. In one embodiment, the composition includes a chemotherapeutic agent. For example, a chemotherapeutic agent can be substantially any agent that exhibits an effect on cancer cells or neoplastic cells of an individual without inhibiting or reducing the tumor-killing effect of MYXV that expresses one or more immunomodulatory transgenes . For example, the chemotherapeutic agent can be (but not limited to) anthracycline, alkylating agent, alkyl sulfonate, aziridine, ethylene imine, methyl imine, nitrogen mustard, nitrosourea, Antibiotics, antimetabolites, folate analogs, purine analogs, pyrimidine analogs, enzymes, podophyllotoxin, platinum-containing agents or cytokines. The chemotherapeutic agent may be a therapeutic agent known to be effective against specific cell types that are cancerous or neoplastic.

The ratio and characteristics of pharmaceutically acceptable diluents can be determined, for example, by the chosen route of administration, compatibility with live viruses, and standard medical practice. In some embodiments, the pharmaceutical composition will be formulated with components that do not significantly impair the biological properties of one or more immunomodulatory transgenic genes, such as BiKE, LIGHT and/or Decorin's MYXV. The pharmaceutical composition can be prepared by a known method for preparing a pharmaceutically acceptable composition suitable for administration to an individual, so that an effective amount of one or more active substances is combined with a pharmaceutically acceptable vehicle A mixture is formed. Suitable vehicles are described in, for example, Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1995). On this basis, the composition may contain MYXV or a solution in which MYXV-containing cells are combined with one or more pharmaceutically acceptable vehicles or diluents, and may be contained in a buffer solution having suitable pH and isotonicity with physiological fluids in.

The pharmaceutical composition can be administered to an individual in various forms depending on the chosen route of administration. The composition of the present invention can be administered orally or parenterally. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, transnasal, intrapulmonary, intrathecal, transrectal and local administration modes. Parenteral administration can be by continuous infusion (for example, intravenous infusion) over a selected period of time.

The pharmaceutical composition can be administered orally, for example with an inert diluent or with a carrier, or it can be enclosed in a hard or soft shell gelatin capsule, or it can be compressed into a lozenge. For oral therapeutic administration, MYXV that exhibits one or more immunomodulatory transgenic genes (such as BiKE, LIGHT and/or Decorin) can be combined with excipients and used as ingestible tablets, buccal tablets, sugar-coated tablets, Capsules, elixirs, suspensions, syrups, powder tablets and similar forms are used.

The MYXV of the present invention or a solution containing cells of the MYXV of the present invention can be prepared in a physiologically suitable buffer. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms, but do not inactivate live viruses. The conventional procedures and ingredients for selecting and preparing suitable formulations are described in, for example, Remington's Pharmaceutical Sciences and The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. The dosage of the pharmaceutical composition to be used depends on the specific disease to be treated, the severity of the disease, individual individual parameters (including age, physical condition, body type and weight), duration of treatment, nature of concurrent therapy (if any), and specific administration It depends on the approach and other similar factors within the knowledge and expertise of the healthcare practitioner. In certain embodiments, the therapeutic virus can be freeze-dried for storage at room temperature. Set

The aspect of the present invention relates to the expression of one or more immunomodulatory transgenic genes, such as MYXV such as BiKE, LIGHT and Decorin, and sets including them. One or more immunomodulatory transgenic genes, such as MYXV such as BiKE, LIGHT and/or Decorin, or a pharmaceutical composition containing MYXV can be packaged as a kit containing instructions for using MYXV, for example. The kit may include any MYXV disclosed herein, such as MYXV including one or more immunomodulatory transgenic genes, one or more reporter genes, one or more non-immunomodulatory transgenic genes, or a combination thereof. In some embodiments, the kit includes MYXV-LIGHT, MYXV-BiKE, MYXV-Decorin, or a combination thereof. In some embodiments, the kit includes MYXV--FLuc-huLIGHT-TdTomato, MYXV-huBiKE-GFP and/or MYXV-mDecorin-GFP. Example Example 1 : The design of the reorganized MYXV structure representing Decorin , LIGHT or BiKE

Build MYXV-mDecorin-GFP

Decorin expression of mouse MYXV virus: Decorin is a member of a family of extracellular matrix proteoglycans of, the extracellular matrix proteoglycan family exist and function (Nemani N, Santo L, Eda H, Cirstea D in stromal and epithelial cells , Mishima Y, Patel C, et al. Role of decorin in multiple myeloma (MM) bone marrow microenvironment. J Bone Miner Res. 2015;30(3):465-70) Cumulative evidence indicates that decorin participates by direct or indirect targeting Signaling molecules for cell growth, survival, cancer metastasis, and angiogenesis (such as targeted chelation) affect the biology of different types of cancer. Decorin can bind to and block the activity of TGF-β, which can be one of the main inhibitory cytokines responsible for immune suppression in the tumor microenvironment. Therefore, the MYXV oncolytic vector expressing Decorin will block TGF-β in the tumor microenvironment and potentially promote a stronger anti-tumor immune response in a wide variety of solid and hematological cancers. In addition, decorin can directly inhibit tumor cell growth and proliferation. For example, adenovirus delivery of decorin to various hematological cancers can counteract tumor formation (Jiang G, Sun C, Li RH, Wei ZP, Zheng JN and Liu YQ. Enhanced antitumor efficacy of a novel oncolytic adenovirus combined with temozolomide in the treatment of melanoma in vivo. J Cancer Res Clin Oncol. 2015;141(1):75-85; Kaliberova LN, Krendelchtchikova V, Harmon DK, Stockard CR, Petersen AS, Markert JM, etc.CRAdRGDflt-IL24 virotherapy in combination with chemotherapy of experimental glioma. Cancer Gene Ther. 2009;16(10):794-805; and Xu W, Neill T, Yang Y, Zebin Hu, Cleveland E, Wu Y, etc. The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer. Gene Ther. 2015;22(3):247-56). These preclinical findings highlight decorin as a potential anticancer target for many types of cancer.

Under the control of poxvirus synthesis early/late promoter (sE/L) and in the wild-type (wt) MYXV strain Lausanne (MYXV-Lau) genome between M135 and M136 genes, MYXV-mDecorin -GFP was constructed by inserting a decorin-expressed cassette containing a murine decorin coding sequence (NM_001190451.2; GenScript) ( Figure 1A ) , which has a C-terminal V5 tag. The expression cassette used to enhance green fluorescent protein (eGFP) is immediately downstream of the inserted decorin expression cassette, and its performance is also driven by the early/late promoter of poxvirus synthesis. eGFP acts as a fluorescent marker for MYXV replication in vitro and in vivo, as MYXV infection can be monitored by real-time imaging of eGFP expression.

In order to generate the MYXV-mDecorin-GFP construct, a gateway system (ThermoFisher Scientific) was first used to construct a recombinant plastid. The upstream and downstream hybridization sequences were amplified by PCR to generate into clones by recombination with the gateway BP of the appropriate pDONR vector. The final recombinant plastid is constructed by recombining the three incoming clones with the target vector in a sequential manner. The Decorin and eGFP expression cassettes were inserted into the MYXV genome by infecting the rabbit kidney13 (RK13) cell line with MYXV-Lau and then transfecting appropriate recombinant plastids. Perform several rounds of lesion purification to obtain a pure stock solution of recombinant virus. Use specific primers for Decorin to confirm the presence of transgenic genes:

AttB4r_mDCN_F: GGGGACAACTTTTCTATACAAAGTTGCCAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAATGAAGGCAACTCTCATCTT ( SEQ ID NO: 1 ).

AttB3r_mDCN_R: GGGGACAACTTTATTATACAAAGTTGTTTAAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACC CTTGTAGTTTCCAAGTTGAA ( SEQ ID NO: 2 ). Purity was confirmed by PCR using appropriate primer sets ( Figure 1B - C ).

By using a mouse monoclonal antibody specific to V5 tag (Invitrogen), several Decorin protein species (40-60 kDa) were detected by Western blot method from the lysate of MYXV-Decorin-infected RK13 cells. ) ( Figure 1D - E ).

Using mink lung epithelial cell line (stably transfected with plasminogen activator inhibitor 1 promoter luciferase construct (MLE/PAI/L cells)) to evaluate the biological activity of MYXV-Decorin-infected RK13 cells Protein and the ability to inhibit the biological activity of TGF-β. Exposure of MLE/PAI/L cells to TGF-β induced a dose-dependent increase in luciferase activity. MLE/PAI/L cells were incubated with a modified medium containing 0.1 ng/mL recombinant TGF-b and recombinant TGF-β mixed with supernatant from RK13 cells. These RK13 cells simulated infection or infection under various conditions MYXV virus strain, and measure the luciferase activity to quantify the biological activity of TGF-β. The supernatant was derived from RK13 cells that: (i) mock infection, (ii) infected with MYXV-GFP (control group without Decorin) at MOI of 10, or (iii) at MOI of 1, 10 or 15 Infect MYXV-mDecorin-GFP. The supernatant from RK13 cells infected with 10 or 15 MYXV-mDecorin-GFP can inhibit the biological activity of TGF-β ( Figure 1F ). The replication ability of the new construct MYXV-mDecorin-GFP in RK13 cells is similar to that of the parental virus MYXV-GFP ( Figure 1G ).

MYXV - BiKE - GFP The construction of

MYXV-Bispecific Natural Killer and Neutrophil Conjugating Molecule (BiKE) (CD138-CD16): BiKE is a molecule containing two single-chain variable fragments (scFv) derived from antibodies through a short peptide linker Join. One scFv arm binds to CD16 on the surface of NK cells and neutrophils, while the other binds to the selected target antigen (in this case, CD138, a hallmark of multiple myeloma (MM) cells). These molecules are designed to form antigen-specific immune synapses between NK cells or neutrophils and tumor cells to trigger enhanced NK/neutrophil cell-mediated killing of tumor targets. Several BiTEs have shown promising results in different murine tumor models (Davis ZB, Vallera DA, Miller JS and Felices M. Natural killer cells unleashed: Checkpoint receptor blockade and BiKE/TriKE utilization in NK-mediated anti-tumor immunotherapy. Semin Immunol. 2017;31:64-75) BiKE constructs extracted and secreted from MYXV can enhance the ability of NK and neutrophils to kill MM cells in TME, and the BiKE technology of virus expression can also be applied to any other cancers , For these cancers, there are cancer-specific cell surface markers.

The anti-CD16 scFv (heavy and light chain variable domains) human Ab sequences were obtained from publicly available sources (AY345160.1 and AY345161.2; Genbank). The anti-CD138 scFv sequence was obtained from a publicly available source (GenBank). In order to form scFv, the anti-CD16 variable region is connected by the (G4S1)3 linker, and the anti-CD138 variable region is connected by the (G4S1)3 linker. The anti-CD16 and anti-CD138 scFv are connected to each other by (G4S1)2 flexible linkers. BiKE is configured with VL(CD138)-VH(CD138)-VH(CD16)-VL(CD16), and includes the signal peptide from the mouse Ig heavy chain and the C-terminal V5 tag ( Figure 2A and SEQ ID NO : 6 ) . The BiKE construct is optimized for human codons and synthesized by Genscript.

Under the control of the poxvirus early/late promoter (sE/L) and in the wild type (wt) MYXV strain Laussane (MYXV-Lau) genome between the M135 and M136 genes, the intergenic position MYXV-BiKE- GFP is constructed by inserting a cassette of BiKE expression containing BiKE coding sequence (GenScript), which has a C-terminal V5 tag. The expression cassette used to enhance green fluorescent protein (eGFP) is immediately downstream of the inserted BiKE expression cassette, and its expression is also driven by the early/late promoter of poxvirus synthesis ( Figure 2B ). eGFP can be used as a fluorescent marker for MYXV replication in vitro and in vivo, as MYXV infection can be monitored by real-time imaging of GFP expression. In order to produce the MYXV-BiKE construct, first use the gateway system (ThermoFisher Scientific) to construct the recombinant plastid. The upstream and downstream hybridization sequences were amplified by PCR to generate into clones by recombination with the gateway BP of the appropriate pDONR vector. The final recombinant plastid is constructed by recombining the three incoming clones with the target vector in a sequential manner. By infecting RK13 cells with MYXV-Lau and then transfecting appropriate recombinant plastids, the BiKE and eGFP expression cassettes were inserted into the MYXV genome. Perform several rounds of lesion purification to obtain a pure stock solution of recombinant virus, and confirm the specificity by PCR using appropriate primer sets:

BiKE_CD16_F TCAGCAAGGACACATCCTCTAA ( SEQ ID NO: 3 )

BiKE_CD16_R TAAGGATCCTCATTGGACTGC ( SEQ ID NO: 4 )

Purity was also confirmed by PCR using appropriate primer sets ( Figure 2C - D ).

By Western blotting method, by detecting the presence of 56 kDa band in RK13 cell lysate from MYXV-BiKE infection, by using mouse monoclonal Ab specific for V5 tag (Invitrogen), both The BiKE performance was confirmed in both the cell lysate and the supernatant ( Figure 2E ). The replication ability of the new construct MYXV-BiKE of RK13 cells is similar to that of the parental virus MYXV-GFP ( Figure 2F ).

SEQ ID NO : 5 provides the nucleotide sequence of the transgenic gene encoding BiKE. SEQ ID NO : 6 provides the amino acid sequence of the transgenic gene encoding BiKE. In SEQ ID NO : 6 , the N-terminal signal sequence is underlined, the linker is in bold, and the C-terminal V5 tag is in italics.

DNA BiKE sequence: ATGAAGAGCCAGACCCAGGTGTTCATCTTCCTGCTGCTGTGCGTGAGCGGCGCCCACGGCGACATCCAGATGACCCAGAGCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGGGTGACCATCAGCTGCAGCGCCAGCCAGGGCATCAACAACTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGGAGCTGCTGATCTACTACACCAGCACCCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCCCGAGGACATCGGCACCTACTACTGCCAGCAGTACAGCAAGCTGCCCAGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGTGGCGGTGGCTCCGGCGGTGGTGGGTCGGGTGGCGGCGGATCTAGCCAGGTGCAGCTGCAGCAGAGCGGCAGCGAGCTGATGATGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGGCCACCGGCTACACCTTCAGCAACTACTGGATCGAGTGGGTGAAGCAGAGGCCCGGCCACGGCCTGGAGTGGATCGGCGAGATCCTGCCCGGCACCGGCAGGACCATCTACAACGAGAAGTTCAAGGGCAAGGCCACCTTCACCGCCGACATCAGCAGCAACACCGTGCAGATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAGGGACTACTACGGCAACTTCTACTACGCCATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGTGGCGGTGGCTCCGGCGGTGGTGGGTCGCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGGACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTAGAGTGGCTGGCACACATTTGGTG GGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAGCAACCAGGTATTCCTCAAAATCGCCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCAAATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCCGGTGGCGGTGGCTCCGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGACACTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGCCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTAA (SEQ ID NO: 5) .

Protein sequence BiKE: MKSQTQVFIFLLLCVSGAHG DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIK GGGGSGGGGSGGGGS SQVQLQQSGSELMMPGASVKISCKATGYTFSNYWIEWVKQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSS GGGGSGGGGS QVTLKESGPGILQPSQTLSLTCSFSGFSLRTSGMGVGWIRQPSGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSSNQVFLKIASVDTADTATYYCAQINPAWFAYWGQGTLVTVSA GGGGSGGGGSGGGGS DTVLTQSPASLAVSLGQRATISCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGIPARFSASGSGTDFTLNIHPVEEEDTATYYCQQSNEDPYTFGGGTKLEIK GKPIPNPLLGLDST (SEQ ID NO: 6).

Build MYXV-FLuc-LIGHTH-TdTomato virus

LIGHT (corresponding to lymphotoxin, presents inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM)-a receptor expressed by T lymphocytes) is a member of the TNF superfamily (gene: TNFSF14) . LIGHT is a type II transmembrane protein that aggregates and binds to three known receptors (herpes simplex virus cell receptor HveA (previously HVEM), lymphotoxin β receptor (Lt-βR) and decoy receptor 3 ( The homotrimeric form of DcR3/TR6). LIGHT is expressed on activated T cells (CD4 ⁺ and CD8 ⁺ types) and immature dendritic cells (DC). Previous studies aimed at triggering T helper 1 (Th1) cytokines The ability of T cell proliferation and secretion distinguishes LIGHT as an immunostimulatory cytokine. Because of its potent immunostimulatory activity in T cells and DCs and its extensive expression of its homologous receptor HveA, LIGHT is an anti-tumor immune response. Promising candidate molecules (Yu P and Fu YX. Targeting tumors with LIGHT to generate metastasis-clearing immunity. Cytokine Growth Factor Rev. 2008;19(3-4):285-94).

It has been shown that the performance of LIGHT in the tumor microenvironment produces a large amount of infiltration of innate T cells, which up-regulates the production of chemokines and the performance of adhesion molecules. The activation of infiltrating T cells may be critical for the positive cancer regression response against immune checkpoint inhibitor (ICI) cancers and the clearance of metastatic cancer sites. (Yu P, Lee Y, Liu W, Chin RK, Wang J, Wang Y et al. Priming of naive T cells inside tumors leads to eradication of established tumors. Nat Immunol. 2004;5(2):141-9; Tang H , Wang Y, Chlewicki LK, Zhang Y, Guo J, Liang W, et al. Facilitating T Cell Infiltration in Tumor Microenvironment Overcomes Resistance to PD-L1 Blockade. Cancer Cell. 2016;29(3):285-96). The LIGHT-mediated activation of LTβR signaling in tumor tissues induces the induction of a variety of chemokines and adhesion molecules, which promotes effective T cell recruitment and T cell activation in tumor tissues. F For example, an adenoviral vector constructed to deliver LIGHT to tumor tissues promotes tumor growth and reduction of cancer metastasis and a stable immune response in mouse tumor models (Yu P and Fu YX. Targeting tumors with LIGHT to generate metastasis-clearing immunity. Cytokine Growth Factor Rev. 2008;19(3-4):285-94).

Based on these immunostimulatory properties, LIGHT is regarded as a promising candidate for cancer immunotherapy. Therefore, the oncolytic MYXV platform that expresses LIGHTH can enhance the anti-cancer immune response, and can, for example, enhance the efficacy of immune checkpoint inhibitors (ICI) in addition to viral therapy.

The cDNA of each gene was used as the starting material to construct recombinant MYXV expressing human or mouse LIGHT/TNFSF14. Human and mouse LIGHT/TNFSF14 cDNA was purchased from GenScript. Transgenic genes can be expressed under the poxvirus synthesis early/late promoter (sE/L), which allows expression only in virus-infected cells. In addition to transgenic genes, reporter genes, such as green fluorescent protein (GFP) or TdTomato genes, can also be used under the poxvirus promoter for rapid selection and purification of recombinant viruses that exhibit transgenic genes. Other reporter genes, such as firefly luciferase (F-Luc), can allow real-time monitoring of virus replication in live animals. Transgenic genes and reporter genes can be inserted between ORF M135 and M136 to maintain the parental wild-type MYXV backbone. Transgenic genes can also be inserted in the background of gene knockout viruses. In this case, the M135 locus was selected to construct a transgenic expression cassette. The final recombination plastid cassette of MYXV contains: the transgenic gene from MYXV, the reporter gene and the gene sequence, and the recombination is carried out at the MYXV.

The construction of recombinant plastids is carried out by Multisite Gateway Pro, which allows the construction of a single plastid by recombination from multiple DNA fragments in bacteria. Generate four pure lines containing different elements to prepare the final recombination cassette. It is: a) element 1, myxoma virus M135 region; b) element 2, human or mouse LIGHT/TNFSF14 cDNA with E/L promoter sequence synthesized by poxvirus; c) element 3, p11 in advanced poxvirus The reporter gene TdTomato under the promoter; d) element 4, the firefly luciferase under the E/L promoter synthesized by the poxvirus, and the myxoma virus M136 gene sequence. In order to prepare the M135KO virus backbone, element 1 was replaced with a partial sequence from the M134 ORF and 50 nt from the M135 ORF. In order to construct the final recombinant plastid cassette, a standard protocol was used to recombine all these four elements with the gateway target vector through the LR recombination reaction. The final recombinant plastids are: a) pDEST M135-136-FLuc-muLIGHT-TdTomato, b) pDEST M135-136-huLIGHT-FLuc-TdTomato, c) pDEST M135KO-Fluc-muLIGHT-TdTomato, and d) pDEST M135KO-FLuc -huLIGHT-TdTomato (Figures 3 and 4). At this stage, before the recombinant virus is produced, Western blot analysis is used to confirm the expression of murine or human LIGHT protein (approximately 26 kDa) under the poxvirus promoter after infection and transfection of plastids in RK13 cells (Figure 5A). The final recombinant plastid encoding the transgenic gene and selection marker together with the flanking sequence was transfected into RK13 cells infected with wild-type MYXV Lausanne. The recombinant virus is isolated from the wild-type virus and continuously purified based on the performance of the selection marker. The performance of transgenic genes was confirmed by western blot analysis and functional analysis (Figure 5B). The 4 viruses are: a) MYXV-FLuc-huLIGHT-TdTomato, b) MYXV-FLuc-mLIGHT-TdTomato, c) MYXV-M135KO-FLuc-huLIGHT-TdTomato, and d) MYXV-M135KO-FLuc-muLIGHT-tdTomato. Example 2 In vitro study using human primary patient samples mixed with multiple myeloma ( MM ) cells

In order to evaluate the susceptibility of primary patient samples mixed with multiple myeloma (MM) cells from 3 drug-resistant patients to MYXV infection, Ficoll-paque plus gradient was used to separate monocytes and remove most of the red blood cells (red blood cell, RBC) for primary unmanipulated peripheral blood (PB) extracts from patients 2, 3, and 4 with different levels of MM cells (CD138 ⁺ ) (summarized in Table 5 ) Purify. Then the suspension of these primary cells were mock-treated (i.e., No virus), or MYXV-BiKE-GFP, MYXV- huLIGHT-FLuc-TdTomato or MYXV-mDecorin-GFP construct incubated together, as shown in Figure 6 - As shown in 13 and Table 6 , under different MOI units, including MOI=10, 1, and 0.1, keep it at 37°C for 1 hour to allow virus adsorption. After that, the mock-treated or MYXV-treated cells were incubated overnight (about 24 hours) at 37°C to allow virus infection. For patient #3, flow cytometry was used to determine the percentage of viral infection at MOI=10, 1.0 and 0.1 ((i.e. use MYXV-FLuc-huLIGHT-TdTomato or MYXV-huBiKE) and survival rate, cell Percentage of apoptosis and cell death ( Figure 6A - C and Figure 8A - C ). In addition, flow cytometry was used to evaluate the survival rate and apoptosis of MM cells in samples from patients exposed to the virus but not infected And the percentage of cell death ( Figures 7A-B and 9A-B ). This allows the measurement not to be directly infected by the virus (that is, not expressing any stereospecific fluorescent protein) but for example by MYXV-activated leukocytes from the same patient sample MM cells among the cells killed by the "off target" method die.

The infection level of MYXV-BiKE-GFP and MYXV-mDecorin-GFP in the cells of patient #4 was used, and the infection level was evaluated by fluorescence microscopy ( Figure 10D and Figure 12D , respectively). Flow cytometry was used to determine the percentage of infected, viable and apoptotic MM cells ( Figure 10A - C and Figure 12A - C ). In addition, flow cytometry was used to evaluate the survival rate, apoptosis and percentage of cell death of these myeloma cells exposed to the virus but not infected ( Figures 11A-B and 12A-B ).

CD138 is used as a marker for multiple myeloma (MM) cells. GFP or TdTomato is used as a marker for MYXV infected cells. Phospholipid binding protein V is used as a marker for apoptotic cells. Near infrared staining is used as a marker for dead cells.

Table 2 and Table 3 summarize the data of patients #3 and #4. Since the number of CD138 ⁺ MM cells in patient 2 is less than 1%, the percentage of MM cell infection and cell death of the virus constructs tested in this patient cannot be determined. The apoptosis and MM cell killing data shown in Table 6 were generated by gating CD138 ⁺ (MM) cells, and the data indicate that all tested virus constructs can effectively infect and kill drugs. MM cells in primary human peripheral blood samples from refractory patients. Strikingly, for patient #3, in all different MOI tests, MYXV constructs exhibiting huLIGHT or BiKE could induce increased killing of MM cells ( Figures 6 , 8 and Table 6 ). For example, 90.8% of cells infected with MYXV expressing huLIGHT at MOI of 10 were killed, 50.1% of cells infected with MYXV expressing huBiKE at MOI of 10 were killed, and 6.51-12.99% of the simulated cells were killed. Healing cells. For patient #4, MYXV expressing BiKE and mDecorin also killed the infected MM cells after virus infection ( Figure 10 , 12 and Table 6 ). For example, kill 35.9% of the cells infected with MYXV expressing BiKE at an MOI of 10, kill 33.1% of the cells infected with MYXV expressing mDecorin at an MOI of 10, and kill 4.85% of the mock-treated cells .

Table 7 shows the data in the MM cell lines by non-infected (i.e., CD138 ⁺ GFP ^- or CD138 ⁺ TdTomato ^-) for gating the killing generated. Compared with the mock-treated cells, the "off-target" killing of this uninfected MM cell of these virus constructs was higher at all MOIs ( Figures 7 and 9 are patient #3, and Table 7 and Figure 11 And 12 are patient #4 and Table 7 ). For example, compared to 5.41% of the simulated treatment cells, 96.12% and 32.3% of uninfected cells from patient 4 were killed in the experiment, where the MYXV construct was added to the culture at an MOI of 10. Table 5 Percentage Hu primary MM cells (CD138 +) of Patient # Patient ID MM cell% 2 PB208485 <1.0 3 PB208482 2.3 4 PB208560 15.0 Table 6 hu -. Primary MM cells were infected, the percentage of apoptosis and cell death (CD138 ^+). Patient #3 ID PB208482 + MYXV MM (CD138 ⁺ GFP ⁺ or CD138 ⁺ TdTomato ⁺ ) infection% under MOI=10, 1.0, 0.1 Phospholipid binding protein V-NIR+ under MOI=10, 1.0, 0.1 Phospholipid binding protein V + near infrared at MOI=10, 1.0, 0.1 Total near infrared under MOI=10, 1.0, 0.1+ MYXV-huBiKE-GFP 48.10, 33.20, 5.90 0.00, 0.089, 0.26 50.10, 32.80, 14.3 50.10, 32.90, 14.56 MYXV-FLuc-huLIGHT -TdTomato 57.60, 15.50, 5.32 34.80, 6.55, 4.95 56.0, 48.80, 31.20 90.80, 55.35, 36.15 Patient #3 ID PB208482 infection% Phospholipid binding protein V-Near Infrared+ Phospholipid binding protein V+Near infrared+ Total NIR+ Analog control of Hu BiKE 0.83 0.91 5.60 6.51 Analog control of huLIGHT 0.98 1.39 11.60 12.99 Patient #4 ID PB208560 + MYXV % Infection of MM (CD138 ⁺ GFP ⁺ or CD138 ⁺ TdTomato ⁺ ) under MOI=10, 1.0, 0.1 Phospholipid binding protein V-near infrared+ at MOI=10, 1.0, 0.1 Under MOI=10, 1.0, 0.1 Phospholipid binding protein V + near infrared + Under MOI=10, 1.0, 0.1, total near infrared + MYXV-huBiKE-GFP 22.10, 5.23, 1.84 6.29, 14.20, 3.00 29.6,0 12.10, 5.53 35.90, 26.30, 8.53 MYXV-mDecorin-GFP 9.97, 3.20, 0.83 14.00, 2.65, 1.89 19.10, 6.94, 5.20 33.10, 9.59, 7.09 Patient #4 ID PB208560 infection% Phospholipid binding protein V-Near Infrared+ Phospholipid binding protein V+Near infrared+ Total NIR+ Analog control of huBiKE 0.86 1.07 3.78 4.85 Analog control of mDecorin 0.63 1.07 3.78 4.85 Table 7 hu uninfected the - primary MM cells (CD138 ⁺⁾ survival rate of infected cell percentage of apoptosis and cell death, such as by of the GFP ^- or tdTomato ^- gating of MM cells were assessed. Patient #3 ID PB208482 + MYXV Phospholipid binding protein V-near infrared+ at MOI=10, 1.0, 0.1 Under MOI=10, 1.0, 0.1 Phospholipid binding protein V + near infrared + Under MOI=10, 1.0, 0.1, total near infrared + MYXV-huBiKE-GFP 16.60, 14.50, 11.90 20.60, 19.70, 17.10 37.20, 34.30, 29.00 MYXV-FLuc-huLIGHT-TdTomato 64.50, 23.30, 20.10 20.30, 58.50, 46.90 84.80, 81.80, 67.00 Patient #3 ID PB208482 Phospholipid binding protein V-Near Infrared+ Phospholipid binding protein V+Near infrared+ Total NIR+ Mock 7.79 11.4 19.200 Patient #4 ID PB208560 + MYXV Phospholipid binding protein V-near infrared+ at MOI=10, 1.0, 0.1 Under MOI=10, 1.0, 0.1 Phospholipid binding protein V + near infrared + Under MOI=10, 1.0, 0.1, total near infrared + MYXV-huBiKE-GFP 9.32, 24.70, 26.10 86.8, 60.1, 31.9 96.12, 84.80, 58.00 MYXV-mDecorin-GFP 15.00, 3.01, 2.06 17.30, 7.41, 5.90 32.30, 10.42, 7.96 Patient #4 ID PB208560 Phospholipid binding protein V-Near Infrared+ Phospholipid binding protein V+Near infrared+ Total NIR+ simulation 1.11 4.30 5.41 Example 3 : Oncolytic virus therapy with myxoma virus ( MYXV ) against multiple myeloma ( MM ) : identification of MYXV constructs suitable for eliminating contaminated cancer cells from primary human samples .

Experiments were performed to identify MYXV constructs and experimental conditions suitable for eliminating contaminated refractory cancer cells from primary human cell samples. Bone marrow or peripheral blood samples are obtained from individuals with hematological cancers such as myeloma, leukemia, or lymphoma. Isolate monocytes (eg via Ficoll-Paque). Samples of monocytes containing cancer cells are treated with the MYXV constructs of the present invention under various conditions (e.g. MOI, incubation time) (e.g., containing different transgenic genes and/or deletions), and as disclosed herein (e.g., via streaming Cytometry, fluorescence microscopy and/or cytotoxicity analysis) to determine the ability of MYXV constructs to kill cancer cells.

The identified constructs and/or experimental conditions can be used to treat individuals. For example, the MYXV construct identified as suitable can be directly administered to the individual (for example, via injection or intravenous infusion), or it can be administered via MYXV adsorbed leukocytes. Example 4 : Oncolytic virus therapy with myxoma virus ( MYXV )

The individual is identified as having a blood cancer (e.g., myeloma, leukemia, or lymphoma). Hematological cancers may be blood cancers including minimal residual disease (MRD) and/or drug-resistant MRD.

Research is conducted as appropriate to identify the MYXV construct of the present invention, which eliminates cancer cells from samples obtained from individuals (eg, peripheral blood or bone marrow samples).

MYXV is administered to the individual (e.g., via injection or infusion). MYXV infects cancer cells in individuals, causing cancer cell killing and anti-cancer immune response. Example 5 : Myxoma virus ( MYXV ) oncolytic virus therapy for autologous transplantation of leukocytes via MYXV - adsorbed .

MYXV is administered to individuals with hematological cancer through autologous transplantation of white blood cells adsorbed by MYXV.

Bone marrow or peripheral blood samples are obtained from individuals with hematological cancers (e.g. myeloma, leukemia, or lymphoma), and monocytes are isolated (e.g. via Ficoll-Paque). Cancer cells can be separated from non-cancer cells (e.g., via FACS or MACS). The MYXV of the present invention is adsorbed to white blood cells (for example, adsorbed at an MOI of about 0.1 to 10 for about one hour). The leukocytes adsorbed by MYXV are administered to the individual via intravenous infusion. MYXV infects cancer cells in individuals, causing cancer cell killing and anti-cancer immune response. Example 6: Oncolytic virus therapy with myxoma virus (MYXV) for allotransplantation of leukocytes adsorbed by MYXV.

MYXV is administered to individuals with hematological cancers (for example, myeloma, leukemia, or lymphoma) through allogeneic transplantation of white blood cells adsorbed by MYXV. The bone marrow or peripheral blood sample is obtained from a donor (e.g., HLA-matched, HLA-mismatched, haploid, or sibling donor, or a combination thereof). Isolate monocytes (eg via Ficoll-Paque). Optionally, cell purification or enrichment is used for specific white blood cell subpopulations (e.g. via FACS or MACS). The MYXV of the present invention is adsorbed to white blood cells (for example, at an MOI of about 0.1 to 10 for about one hour). The leukocytes adsorbed by MYXV are administered to the individual via intravenous infusion. MYXV infects cancer cells in individuals, causing cancer cell killing and anti-cancer immune response. Example 7: In the minimal residual disease (the MRD) of potential Vk * MYC mice immunized with autologous MYXV ex vivo binding of virus to target therapy to eliminate vivo and resistance disseminated MM.

Two C57BL/6-derived VK*MYC cell lines were used in in vivo experiments: bortezomib resistant VK12598 and multiple drug resistant VK12653. First, evaluate the susceptibility of these two VK*MYC cell lines to MYXV binding and infection.

MYXV binding to VK12598 and VK12653 in vitro study : For the binding experiment, MYXV-M093L-Venus virus (a fusion containing the fluorescent protein Venus at the amino end of M093L) was used at a magnification of infection (MOI) of 10. In short, VK12598 or VK12653 was newly separated from BM (or with newly thawed BM) and incubated with MYXV-M093L-Venus at 4°C for 1 hour to allow virus binding. Unbound virus is removed by washing virus-adsorbed cells twice. Use flow cytometry to quantify the level of viral particle binding. To analyze virus infection, cells were incubated with reporter MYXV-GFP (E/L)/TdTomato(L) at MOI=10 at 37°C for 1 hour to allow virus adsorption. The cells were incubated at 37°C overnight to allow virus infection. MYXV effectively binds to two cell lines ( Figure 14A and 15A ). In addition, MYXV effectively infects two cell lines ( Figure 14B-C and 15B ).

In vivo study using the VK12598 cell line : In the first in vivo experiment, C57BL/6 mice were pre-inoculated with VK12598 cells (for example, 1×10 ⁶ cells per mouse). Four weeks after MM cell implantation, the mice were bled and M spikes were measured. Mice were separated according to the level of M peak (for example, 0, low=0.1, medium=0.2, high=0.6) ( Figure 16A , top panel). The mice were then treated as follows: without C57BL/6 BM transplantation (group I), C57BL/6 BM cells alone (group II), MYXV-M135KO-GFP alone (group III), and MYXV-M135KOK-GFP in vitro Treatment of C57BL/6 BM (Group IV) ( Figure 16A , bottom panel). Mice and Representative spikes from having a higher M (0.6) of the Group II ^- FIG. 16B shows the percentage (CD138 ⁺ B220) from representative mice of group I has a lower peak M (0.1) of the MM MM percentage of (CD138 ⁺ B220 ^-). Figure 16C shows the M spike of the only survivor from Group IV, which shows complete regression of MM, in which no M spike band was detected on the 8, 29 and 37 days after transplantation. These data indicate that transplantation of isolated MYXV-treated bone marrow can induce MM regression. In summary, these data also indicate that the cohort therapy in this first experiment started too late in disease progression, and in fact, the viral therapy should be started earlier in this model (for example, less than 1 week after MM implantation and Not 4 weeks after MM implantation). Although regression of MM can occur even at this late intervention time, early initiation of viral therapy (e.g., in a group of mice that are not so close to the point of death or termination) may allow for improved evaluation of viral technology.

In additional trials, MYXV was tested in combination with other therapeutic agents (such as the SMAC mimic LC161). VK12598 cancer cells were implanted, M spikes were quantified in 1 to 4 weeks, and mice were treated with cyclophosphamide to induce a transient complete response (CR), which could last for 1 month. One or two weeks after cyclophosphamide, the mice were transplanted with BM+MYXV or PBMC+MYXV (for example, MYXV exhibiting the immunomodulatory transgenic gene as disclosed herein) to test whether the viral therapy can be extended or completed The partial subsidence initiated by cyclophosphamide. In this case, the ability of MYXV to eliminate MM minimal residual disease (MRD) as defined by the disease that is functionally resistant to this chemotherapy is studied. The ability of MYXV to eliminate multiple drug-resistant VK12653 cell lines as a single therapy or as a combination therapy is also studied.

Although the present invention has been described with emphasis on specific examples, it will be obvious to those skilled in the art that variations of specific compositions can be used, and it is intended that the present invention can be implemented in ways other than those specifically described herein. The features, characteristics, compounds or examples described in conjunction with specific aspects, embodiments or examples of the present invention should be understood as applicable to any other aspects, embodiments or examples of the present invention. Therefore, the present invention includes all modifications within the spirit and scope of the present invention as defined by the scope of the following patent applications. We therefore claim that the present invention falls within the scope and spirit of these patent applications.

The novel features of certain embodiments of the present invention are described in detail in the scope of the attached patent application. A better understanding of the features and advantages of the present invention will be obtained with reference to the following detailed description illustrating exemplary embodiments using the principles of the present invention and the accompanying drawings:

Figures 1A - 1G show the construction of MYXV-mDecorin-GFP. Figure 1A is a schematic diagram of the MYXV genome and the insertion site of the cassette expressing Decorin and eGFP. Both transgenic genes are expressed under the poxvirus synthesis early/late promoter (sE/L). Figure 1B shows the PCR analysis of genomic viral DNA from MYXV-mDecorin-GFP pure line using oligonucleotide primers to confirm the presence of Decorin (Figure 1-5), and Figure 1C shows the proper insertion (intergenic region M135-M1356) ). Ribbon 1 is the DNA from MYXV-Lau, ribbons 2-6: MYXV-mDecorin-GFP pure line, and M represents the DNA step of known size. Figure 1D shows the Western blot analysis of the cell lysate, and Figure 1E shows the Western blot analysis of the supernatant. The cell lysate and supernatant are from MYXV-mDecorin-GFP infected RK13 24 hours after infection cell. Figure 1F shows the luciferase activity in MLE/PAI/LUC cells incubated with modified medium derived from RK13 cells infected with MYXV encoding decorin or GFP. Figure 1G shows a single-step growth analysis of recombinant MYXV-Decorin relative to MYXV-GFP.

Figure 2A - 2F show the construction of MYXV-BiKE. Figure 2A shows a schematic diagram of the structure of human CD138 targeting BiKE. Figure 2B is a schematic diagram of the MYXV genome and the insertion site of the cassette expressing BiKE and eGFP. Both transgenic genes are expressed under the poxvirus synthesis early/late promoter (sE/L). Figure 2C shows the PCR analysis of genomic viral DNA from MYXV-BiKE clones using oligonucleotide primers to confirm the presence of the BiKE cassette (Figures 1-4), and Figure 2D shows the proper insertion (intergenic region M135-M1356) ). Ribbon 1 is the DNA from MYXV-Lau, ribbon 2-4: MYXV-BiKE pure line, M represents DNA step of known size. Figure 2E shows Western blot analysis of cell lysates and supernatants from MYXV-Decorin-infected RK13 cells 24 hours after infection. Figure 2F shows the single-step growth analysis of recombinant MYXV-BIKE relative to MYXV-GFP.

Fig. 3 is a plasmid map showing wild-type (wt) myxoma virus produced by human and murine LIGHT. It should be noted that this virus has a wild-type MYXV backbone, because the LIGHT transgenic gene is inserted into the intergenic locus between the M135R and M136R genes of the virus, and therefore there is no knockout or destruction of M135R or M136R genes.

Figure 4 is a plastid map showing the expression of M135KO myxoma virus produced by human and murine LIGHT. It should be noted that this virus has a gene knockout of the viral M135R gene.

Figure 5A shows the Western blot analysis of human and murine LIGHT performance from recombinant plastids under the poxvirus sE/L promoter.

Figure 5B shows a Western blot analysis of the human and murine LIGHT performance from recombinant MYXV.

Figures 6A - 6C show that MYXV-FLuc-huLIGHT-TdTomato effectively infects and induces the killing of MM cells in primary human samples from patient #3 with direct multiple myeloma (MM). Figure 6A shows the infection (TdTomato+), survival rate (near-infrared-), apoptosis (phospholipid binding protein V+) and cell killing of MM cells (CD138 ⁺ ) and simulated treatment (ie, no MYXV added) samples (Near infrared +) situation. Figure 6B shows the CD138 ⁺ MYXV-FLuc-huLIGHT-TdTomato infection (TdTomato+) under three different MOIs. Figure 6C shows the apoptosis and cell death of CD138 ⁺ cells induced by MYXV-FLuc-huLIGHT-TdTomato.

7A and 7B show MM cells (i.e. TdTomato ^-) cells from the patient with multiple myeloma (MM) # 3 of primary human samples killing of uninfected case, the multiple myeloma (MM) The cells were exposed to MYXV-FLuc-huLIGHT-TdTomato. 7A shows simulated MM cell sample treatment (i.e., without addition MYXV) after 24 hours of uninfected (i.e., CD138 ⁺ TdTomato ^-) survival (near infrared -), apoptosis (annexin V + ) And cell killing (near infrared +) situation. Arrows indicate gate control of TdTomato-cells. Figure 7B shows the percentage of apoptosis and cell death of CD138 ⁺ TdTomato ^- cells 24 hours after treatment with virus.

Figures 8A - 8C show that MYXV-huBiKE-GFP effectively infects and induces killing of primary human samples from patient #3 with multiple myeloma (MM). Figure 8A shows the infection (GFP+), survival rate (near-infrared-), apoptosis (phospholipid binding protein V+) and cell killing of MM cells (CD138 ⁺ ) of samples subjected to simulated treatment (ie, no MYXV added) (Near infrared +) situation. Figure 8B shows the CD138 ⁺ MYXV-huBiKE-GFP infection at three different MOIs. Figure 8C shows the apoptosis and cell death of CD138 ⁺ cells induced by MYXV-huBiKE-GFP.

9A and 9B shows a patient from the rear MYXV-huBiKE-GFP # Primary treatment of multiple myeloma in human samples of uninfected of 3 (MM) cells (i.e. GFP ^-) killing conditions. 9A shows MM cells (i.e., CD138 ⁺ GFP ^-) mock-treated samples were after 24 hours (i.e., No MYXV) the survival was not infected (near infrared -), apoptosis (annexin V +) And cell killing (near infrared +) situation. The arrow indicates the gating of GFP-cells. Figure 9B shows the percentage of apoptosis and cell death of uninfected MM cells, which are CD138 ⁺ GFP ^- 24 hours after infection.

Figures 10A - 10D show that MYXV-huBiKE-GFP effectively infects and induces killing multiple myeloma (MM) cells from the primary human sample of patient #4. Figure 10A shows the survival rate (near-infrared-), infection (GFP+), apoptosis (phospholipid binding protein V+) and cell killing of MM cells (CD138 ⁺ ) after 24 hours of simulated treatment (ie, no MYXV added) (Near infrared +) situation. Figure 10B shows the CD138 ⁺ MYXV-huBiKE-GFP infection at three different MOIs. Figure 10C shows the apoptosis and cell death of CD138 ⁺ cells induced by MYXV-huBiKE-GFP. Figure 10D shows a fluorescence micrograph 24 hours after infection.

11A and 11B show from a patient after the MYXV-huBiKE-GFP # treatment of multiple myeloma, non-infected primary human samples 4 (MM) cells (i.e. GFP ^-) killing conditions. 11A shows MM cells (i.e., CD138 ⁺ GFP ^-) samples were mock-treated (i.e., no addition MYXV) after 24 hours of survival uninfected (near infrared -), apoptosis (annexin V +) And cell killing (near infrared +) situation. The arrow indicates the gating of GFP- (uninfected). Figure 11B shows the percentage of apoptosis and cell death of uninfected CD138 ⁺ GFP ^- cells 24 hours after treatment with virus.

Figures 12A - 12D show that MYXV-mDecorin-GFP effectively infects and induces the killing of multiple myeloma (MM) cells in a primary human sample from patient #4. Figure 12A shows the infection (GFP+), apoptosis (phospholipid binding protein V+) and cell killing (near infrared+) of MM cells (CD138 ⁺ ) after 24 hours of simulated treatment (ie, no MYXV added). Figure 12B shows the CD138 ⁺ MYXV-mDecorin-GFP infection at three different MOIs. Figure 12C shows the survival rate, apoptosis and cell death of CD138 ⁺ cells induced by MYXV-mDecorin-GFP. Figure 12D shows a fluorescence micrograph 24 hours after infection.

13A and 13B shows a patient from MYXV-mDecorin-GFP # treatment of multiple myeloma (MM) 4 primary human samples of non-infected cells (i.e., GFP ^-) killing conditions. 13A shows a simulated MM cell sample 24 hours after treatment (i.e., without addition MYXV) of not infected (i.e., CD138 ⁺ GFP ^-) survival (near infrared -), apoptosis (annexin V + ) And cell killing (near infrared +) situation. The arrow indicates the gating of GFP-cells (uninfected). Figure 13B shows the percentage of apoptosis and cell death of uninfected CD138 ⁺ GFP ^- cells 24 hours after infection.

Figures 14A - 14C show that the BOR-anti-VK12598 cell line is sensitive to MYXV. Figure 14A shows the binding of MYXV to the VK12598 cell line (Venus+). Figure 14B shows the effective infection of the VK12598 cell line obtained by fluorescence microscopy. Figure 14C shows the effective infection of the VK12598 cell line obtained by flow cytometry.

Figures 15A and 15B show the MYXV binding and infection status of the multi-drug resistant VK12653 cell line. Figure 15A shows the binding of MYXV to the VK12653 cell line (Venus+). Figure 15B shows the effective infection of the VK12598 cell line obtained by fluorescence microscopy and flow cytometry.

Figures 16A - 16C show the ex vivo therapy of myxoma virus to treat pre-existing multiple myeloma cancer in autologous transplant recipients. Figure 16A shows the Western blot method, which provides M spikes of mice four weeks after VK12598 cell implantation (upper image) and four experimental groups (lower image). The level of the M spike four weeks after MM cell implantation. FIG. 16B shows a representative mouse (the mock-treated mice) M having a lower peak (0.1) of the MM cells (CD138 ⁺ B220 ^-) percentage, and having a high peak M (0.6) of the bone marrow by a representative mice in MM cells (CD138 ⁺ B220 ^-) percentage. Figure 16C shows that in mice treated with bone marrow (which had been treated with MYXV-M135KO-GFP ex vivo), M spike bands were not detected on the 8, 29 and 37 days after transplantation.

Claims (119)

A myxoma virus (MYXV) containing one or more immunomodulatory transgenic genes, wherein the one or more immunomodulatory transgenic genes encode BiKE (bispecific natural killer and neutrophil conjugating molecule), LIGHT (lymphatic Toxins, present inducible performance, and compete with HSV glycoprotein D for herpes virus entry mediator (HVEM)-the receptor expressed by T lymphocytes), Decorin or a combination thereof. The myxoma virus of claim 1, wherein the myxoma virus comprises MYXV-BiKE. The myxoma virus of claim 1 or claim 2, wherein the BiKE binds to an antigen present on natural killer cells, neutrophils, or a combination thereof. The myxoma virus according to any one of claims 1 to 3, wherein the BiKE binds to an antigen present on blood cancer cells. The myxoma virus according to any one of claims 1 to 4, wherein the BiKE binds to an antigen present on myeloma cells. The myxoma virus of any one of claims 1 to 5, wherein the BiKE binds to an antigen present on leukemia cells. The myxoma virus according to any one of claims 1 to 6, wherein the BiKE binds to an antigen present on lymphoma cells. The myxoma virus according to any one of claims 1 to 7, wherein the BiKE binds to CD16. The myxoma virus according to any one of claims 1 to 8, wherein the BiKE binds CD138. The myxoma virus according to any one of claims 1 to 9, wherein the BiKE comprises one or more single-chain variable fragments (scFv). The myxoma virus according to any one of claims 1 to 10, wherein the BiKE comprises one or more humanized single-chain variable fragments (scFv). The myxoma virus according to any one of claims 1 to 11, wherein the BiKE comprises a sequence that is at least 70% identical to any one of SEQ ID NO: 6 or 16-31. The myxoma virus according to any one of claims 1 to 12, wherein the BiKE is between the M135 and M136 open reading frames of the myxoma virus genome. The myxoma virus according to any one of claims 1 to 13, wherein the myxoma virus comprises MYXV-huBiKE-GFP. The myxoma virus of claim 1, wherein the myxoma virus comprises MYXV-LIGHT. The myxoma virus of claim 15, wherein the LIGHT comprises a sequence derived from human LIGHT. Such as the myxoma virus of claim 15 or claim 16, wherein the LIGHT comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 13-15. The myxoma virus according to any one of claims 15 to 17, wherein the LIGHT is between the M135 and M136 open reading frames of the myxoma virus genome. The myxoma virus according to claim 1 or any one of 15 to 18, wherein the myxoma virus comprises MYXV-FLuc-huLIGHT-TdTomato. Such as the myxoma virus of claim 1, wherein the myxoma virus contains MYXV-Decorin. Such as the myxoma virus of claim 20, wherein the Decorin comprises a sequence from human Decorin. The myxoma virus of claim 20, wherein the Decorin comprises a sequence from mouse Decorin. The myxoma virus according to any one of claims 20 to 22, wherein the Decorin comprises a sequence that is at least 70% identical to any one of SEQ ID NO: 7-12. The myxoma virus of any one of claims 20 to 23, wherein the Decorin is between the M135 and M136 open reading frames of the myxoma virus genome. The myxoma virus according to claim 16, wherein the myxoma virus contains MYXV-mDecorin-GFP. Such as the myxoma virus of any one of claims 1 to 25, which further comprises a reporter gene. Such as the myxoma virus of claim 26, wherein the reporter gene is a fluorescent protein. Such as the myxoma virus of claim 27, wherein the reporter gene is a luminescent substrate or an enzyme. The myxoma virus according to any one of claims 1 to 28, which further comprises a deletion in the myxoma virus genome. The myxoma virus according to any one of claims 1 to 29, wherein the myxoma virus comprises deletion or destruction of one or more genes selected from the group consisting of: M001R, M002R, M003.1R, M003.2R, M004 .1R, M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M -T2, M-T4, M-T5, M-T7 and SOD. The myxoma virus according to any one of claims 1 to 30, wherein the myxoma virus comprises a deletion of M135. A composition comprising the myxoma virus according to any one of claims 1 to 31 and a pharmaceutically acceptable carrier. A method for treating blood cancer in an individual in need, which comprises administering to the individual a myxoma virus containing one or more immunomodulatory transgenic genes. The method of claim 33, wherein the one or more immunomodulatory transgenic genes comprise BiKE, LIGHT, Decorin or a combination thereof. Such as the method of claim 33 or claim 34, wherein the myxoma virus comprises MYXV-BiKE. The method of claim 34 or claim 35, wherein the BiKE binds to an antigen present on natural killer cells, neutrophils, or a combination thereof. The method according to any one of claims 34 to 36, wherein the BiKE binds to an antigen present on blood cancer cells. The method according to any one of claims 34 to 37, wherein the BiKE binds to an antigen present on myeloma cells. The method according to any one of claims 34 to 37, wherein the BiKE binds to an antigen present on leukemia cells. The method according to any one of claims 34 to 37, wherein the BiKE binds to an antigen present on lymphoma cells. The method of any one of claims 34 to 40, wherein the BiKE binds to CD16. The method of any one of claims 34 to 41, wherein the BiKE binds CD138. The method according to any one of claims 34 to 42, wherein the BiKE comprises one or more single chain variable fragments (scFv). The method according to any one of claims 34 to 43, wherein the BiKE comprises one or more humanized single-chain variable fragments (scFv). The method according to any one of claims 34 to 44, wherein the BiKE comprises a sequence that is at least 70% identical to any one of SEQ ID NO: 6 or 16-31. The method of any one of claims 34 to 45, wherein the BiKE is between the M135 and M136 open reading frames of the myxoma virus genome. The method according to any one of claims 34 to 46, wherein the myxoma virus comprises MYXV-huBiKE-GFP. Such as the method of claim 33 or claim 34, wherein the myxoma virus comprises MYXV-LIGHT. Such as the method of claim 32 or claim 48, wherein the LIGHT includes a sequence from a human LIGHT. The method according to any one of claim 34, 48 or 49, wherein the LIGHT comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 13-15. The method according to any one of claim 34 or 48 to 50, wherein the LIGHT is between the M135 and M136 open reading frames of the myxoma virus genome. The method according to any one of claim 34 or 48 to 51, wherein the myxoma virus comprises MYXV-FLuc-huLIGHT-TdTomato. Such as the method of claim 33 or claim 34, wherein the myxoma virus comprises MYXV-Decorin. Such as the method of claim 34 or claim 53, wherein the Decorin includes a sequence from a human Decorin. Such as the method of claim 34 or claim 53, wherein the Decorin comprises a sequence from mouse Decorin. The method of any one of claims 53 to 55, wherein the Decorin comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 7-12. The method according to any one of claims 53 to 56, wherein the Decorin is between the M135 and M136 open reading frames of the myxoma virus genome. The method according to any one of claims 53 to 57, wherein the myxoma virus comprises MYXV-mDecorin-GFP. The method according to any one of claims 33 to 58, wherein the myxoma virus further comprises a reporter gene. The method of claim 59, wherein the reporter gene is a fluorescent protein. The method of claim 59, wherein the reporter gene is a luminescent substrate or an enzyme. The method according to any one of claims 33 to 61, wherein the myxoma virus further comprises a deletion in the myxoma virus genome. The method according to any one of claim 33 to 62, wherein the myxoma virus comprises deletion or destruction of one or more genes selected from the group consisting of: M001R, M002R, M003.1R, M003.2R, M004.1R , M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2 , M-T4, M-T5, M-T7 and SOD. The method according to any one of claims 33 to 63, wherein the myxoma virus comprises a deletion of M135. The method according to any one of claims 33 to 64, wherein the individual is a human. The method according to any one of claims 33 to 65, wherein the myxoma virus is capable of infecting cells with defective innate antiviral response. The method according to any one of claims 33 to 66, wherein the myxoma virus can infect cancer cells. The method according to any one of claims 33 to 67, wherein the blood cancer is myeloma, leukemia or lymphoma. The method according to any one of claims 33 to 67, wherein the blood cancer is multiple myeloma. A method for treating hematological cancer in an individual in need, which comprises administering white blood cells to the individual, wherein the white blood cells comprise myxoma virus containing one or more immunomodulatory transgenic genes. The method of claim 70, further comprising adsorbing the myxoma virus to the surface of the white blood cell in vitro. The method of claim 71, wherein adsorbing the myxoma virus to the surface of the white blood cell comprises exposing the white blood cell to the myxoma virus under conditions that permit the myxoma virus to bind to the surface of the white blood cell. The method of claim 72, wherein the myxoma virus is exposed to the white blood cells for at least five minutes. The method of claim 73, wherein the myxoma virus is exposed to the white blood cells for about one hour. The method of any one of claims 72 to 74, wherein the myxoma virus is exposed to the white blood cells at a multiplicity of infection (MOI) between about 0.001 and 1000. The method of claim 75, wherein the myxoma virus is exposed to the white blood cells at a magnification of infection (MOI) between about 0.1 and 10. The method of any one of claims 70 to 76, wherein the white blood cells are obtained from peripheral blood. The method of any one of claims 70 to 76, wherein the white blood cells are obtained from bone marrow. The method according to any one of claims 70 to 77, wherein the white blood cells are peripheral blood mononuclear cells. The method of any one of claims 70 to 79, wherein the white blood cells are obtained from the individual. The method according to any one of claims 70 to 79, wherein the white blood cells are obtained from a donor that is HLA-matched, HLA-unmatched, haploid or a combination thereof with respect to the individual. The method according to any one of claims 70 to 81, wherein the one or more immunomodulatory transgenic genes comprise BiKE, LIGHT, Decorin or a combination thereof. The method according to any one of claims 70 to 82, wherein the myxoma virus comprises MYXV-BiKE. The method of claim 82 or claim 83, wherein the BiKE binds to an antigen present on natural killer cells, neutrophils, or a combination thereof. The method according to any one of claims 82 to 84, wherein the BiKE binds to an antigen present on blood cancer cells. The method according to any one of claims 82 to 85, wherein the BiKE binds to an antigen present on myeloma cells, leukemia cells, or lymphoma cells. The method of any one of claims 82 to 86, wherein the BiKE is bound to CD16. The method according to any one of claims 82 to 87, wherein the BiKE binds CD138. The method according to any one of claims 82 to 88, wherein the BiKE comprises one or more single-chain variable fragments (scFv). The method according to any one of claims 82 to 89, wherein the BiKE comprises one or more humanized single-chain variable fragments (scFv). The method according to any one of claims 82 to 90, wherein the BiKE comprises a sequence that is at least 70% identical to any one of SEQ ID NO: 6 or 16-31. The method according to any one of claims 82 to 91, wherein the BiKE is between the M135 and M136 open reading frames of the myxoma virus genome. The method according to any one of claims 82 to 92, wherein the myxoma virus comprises MYXV-huBiKE-GFP. The method according to any one of claims 70 to 82, wherein the myxoma virus comprises MYXV-LIGHT. Such as the method of claim 82 or claim 94, wherein the LIGHT includes a sequence from a human LIGHT. The method according to claim 82 or any one of 94 to 95, wherein the LIGHT comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 13-15. The method according to claim 82 or any one of 94 to 96, wherein the LIGHT is between the M135 and M136 open reading frames of the myxoma virus genome. The method according to any one of claim 82 or 94 to 97, wherein the myxoma virus comprises MYXV-FLuc-huLIGHT-TdTomato. The method according to any one of claims 70 to 82, wherein the myxoma virus comprises MYXV-Decorin. Such as the method of claim 82 or claim 99, wherein the Decorin includes a sequence from a human Decorin. Such as the method of claim 82 or claim 99, wherein the Decorin comprises a sequence from mouse Decorin. The method according to claim 82 or any one of 99 to 101, wherein the Decorin comprises a sequence that is at least 70% identical to any one of SEQ ID NOs: 7-12. The method according to any one of claim 82 or 99 to 102, wherein the Decorin is between the M135 and M136 open reading frames of the myxoma virus genome. The method according to any one of claim 82 or 99 to 103, wherein the myxoma virus comprises MYXV-mDecorin-GFP. The method according to any one of claims 70 to 104, wherein the myxoma virus further comprises a reporter gene. Such as the method of claim 105, wherein the reporter gene is a fluorescent protein. The method of claim 105, wherein the reporter gene is a luminescent substrate or an enzyme. The method according to any one of claims 70 to 107, wherein the myxoma virus further comprises a deletion in the myxoma virus genome. The method according to any one of claims 70 to 108, wherein the myxoma virus comprises deletion or destruction of one or more genes selected from the group consisting of: M001R, M002R, M003.1R, M003.2R, M004.1R , M004R, M005R, M006R, M007R, M008.1R, M008R, M009L, M013, M036L, M063L, M11L, M128L, M131R, M135R, M136R, M141R, M148R, M151R, M152R, M153R, M154L, M156R, M-T2 , M-T4, M-T5, M-T7 and SOD. The method according to any one of claims 70 to 109, wherein the myxoma virus comprises a deletion of M135. The method according to any one of claims 70 to 110, wherein the individual is a human individual. The method of any one of claims 70 to 111, wherein the individual has or is suspected of having cancer. The method according to any one of claims 70 to 112, wherein the myxoma virus can infect cells with defective innate antiviral response. The method according to any one of claims 70 to 113, wherein the blood cancer is myeloma, leukemia or lymphoma. The method according to any one of claims 70 to 114, wherein the blood cancer is multiple myeloma. The method according to any one of claims 70 to 115, wherein the white blood cells are administered in the form of a pharmaceutical composition. The method according to any one of claims 70 to 116, wherein the white blood cells are administered systemically. The method according to any one of claims 70 to 116, wherein the white blood cells are administered parenterally. The method of any one of claims 70 to 116, wherein the white blood cells are administered by infusion.

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